Anti-TNF-α antibodies and their uses

ABSTRACT

The present disclosure relates to antibodies directed to the tumor necrosis factor alpha (“TNF-α”) and uses of such antibodies, for example, to treat diseases associated with the activity and/or overproduction of TNF-α.

1. CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.provisional application No. 61/170,053, filed Apr. 16, 2009, thecontents of which are incorporated herein by reference in theirentirety.

2. REFERENCE TO SEQUENCE LISTING

The Sequence Listing concurrently submitted herewith under 37 CFR §1.821in a computer readable form (CRF) via EFS-Web as file name 381493US.txtis incorporated herein by reference. The electronic copy of the SequenceListing was created on Apr. 14, 2010, with a file size of 109,937 bytes.

3. FIELD OF THE INVENTION

The present invention relates to anti-TNF-α antibodies, pharmaceuticalcompositions comprising anti-TNF-α antibodies, and therapeutic uses ofsuch antibodies.

4. BACKGROUND

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine thatis released by and interacts with cells of the immune system. TNF-α hasbeen shown to be upregulated in a number of human diseases, includingchronic diseases such as rheumatoid arthritis, Crohn's disease,ulcerative colitis and multiple sclerosis. For example, elevated levelsof TNF-α are found in the synovial fluid of rheumatoid arthritispatients and play an important role in both the pathologic inflammationand the joint destruction that are hallmarks of rheumatoid arthritis.

Human TNF-α is a 17 kDa protein, and the active form exists as ahomotrimer (Pennica et al., 1984, Nature 312:724-729; Davis et al.,1987, Biochemistry 26:1322-1326; Jones et al., 1989, Nature338:225-228). TNF-α exerts its biological effects through interactionwith two structurally related but functionally distinct cell surfacereceptors, p55 and p75, that are co-expressed on most cell types(Loetscher et al., 1990, Cell 61:351-9; Smith et al., 1990, Science248(4958):1019-23). p55 is also known as p55R; p55TNFR; CD120a; TNFR I;TNFR 1 and TNFRSFIa. p75 is also known as p75R; p75TNFR; CD120b; TNFRII; TNFR 2 and TNFRSFIb. Both receptors are also proteolyticallyreleased as soluble molecules capable of binding TNF-α.

Inhibition of TNF-α activity as a method of treating disease, inparticular, rheumatoid arthritis, has been achieved by a number ofdifferent means using inhibitors such as antibodies and solublereceptors. Examples include etanercept, marketed by Immunex Corporationas ENBREL® which is a recombinant fusion protein comprising two p75soluble TNF-receptor domains linked to the Fc portion of a humanimmunoglobulin. Infliximab, marketed by Centocor Corporation asREMICADE®, is a chimeric antibody having murine anti-TNF-α variabledomains and human IgG₁ constant domains. Other inhibitors includeengineered TNF-α molecules which form trimers with native TNF-α andprevent receptor binding (Steed et al., 2003, Science 301:1895-1898; WO03/033720; WO 01/64889). These current methods of inhibiting TNF-αactivity block binding of TNF-α to both the p55 and p75 receptors (See,for example, Mease, 2005, Expert Opin. Biol. Therapy 5(11):1491-1504).Adalimumab, marketed by Abbott Laboratories as HUMIRA®, is arecombinant, fully human anti-TNF-α antibody (Tussirot and Wendling,2004, Expert Opin. Pharmacother. 5:581-594). Adalimumab bindsspecifically to TNF-α and blocks its interaction with the p55 and p75cell surface TNF-α receptors. Adalimumab also lyses surface TNF-αexpressing cells in vitro via complement-dependent cytotoxicity (“CDC”)and antibody-dependent cell-mediated cytotoxicity (“ADCC”). Adalimumabdoes not bind or inactivate lymphotoxin (TNF-β). Adalimumab alsomodulates biological responses that are induced or regulated by TNF,including changes in the levels of adhesion molecules responsible forleukocyte migration (ELAM-1, VCAM-1, and ICAM-1 with an IC₅₀ of1-2×10⁻¹⁰ M).

Despite being a human antibody, Adalimumab can elicit an immune responsewhen administered to humans. Such an immune response can result in animmune complex-mediated clearance of the antibodies or fragments fromthe circulation and make repeated administration unsuitable for therapy,thereby reducing the therapeutic benefit to the patient and limiting thereadministration of the antibody.

Accordingly, there is a need to provide improved anti-TNF-α antibodiesor fragments that overcome one more of these problems, for example, bygenerating variants with higher affinity than Adalimumab that can beadministered at reduced dosages or variants with reduced immunogenicityas compared to Adalimumab.

Citation or identification of any reference in Section 4 or in any othersection of this application shall not be construed as an admission thatsuch reference is available as prior art to the present disclosure.

5. SUMMARY

The present disclosure relates to variants of the anti-TNF-α antibodyD2E7 with improved binding to TNF-α and/or reduced immunogenicity ascompared to D2E7. D2E7 has three heavy chain CDRs, referred to herein(in amino- to carboxy-terminal order) as CDR-H1 (SEQ ID NO:5), CDR-H2(SEQ ID NO:6), and CDR-H3 (SEQ ID NO:7), and three light chain CDRs,referred to herein (in amino- to carboxy-terminal order) as CDR-L1 (SEQID NO:8), CDR-L2 (SEQ ID NO:9), and CDR-L3 (SEQ ID NO:10). Theanti-TNF-αantibodies and anti-TNF-α binding fragments of the disclosuregenerally have at least one amino acid substitution in at least one CDRas compared to D2E7.

In certain aspects, at least one amino acid substitution or combinationof substitutions is selected from FIG. 17, FIG. 18 and/or FIG. 31.Further mutations (including substitutions, deletions or insertions) canbe selected from one or more of FIGS. 19-31.

In certain aspects, the present disclosure relates to variants of theanti-TNF-α antibody D2E7 with improved binding properties, e.g.,improved affinity, to TNF-α as compared to D2E7. In specificembodiments, the antibodies of the disclosure have a greater affinitythan D2E7 towards TNF-α, for example improved K_(D) as measured byBIAcore and/or improved affinity as measured by competition ELISA.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments include at least one substitution selected from S3K in CDR-L2(SEQ ID NO:9), S3R in CDR-L2 (SEQ ID NO:9), S3N in CDR-L2 (SEQ ID NO:9),T4H in CDR-L2 (SEQ ID NO:9), T4Q in CDR-L2 (SEQ ID NO:9), T4V in CDR-L2(SEQ ID NO:9), T4F in CDR-L2 (SEQ ID NO:9), T4W in CDR-L2 (SEQ ID NO:9),T4Y in CDR-L2 (SEQ ID NO:9); L5R in CDR-L2 (SEQ ID NO:9), L5K in CDR-L2(SEQ ID NO:9), Q6K in CDR-L2 (SEQ ID NO:9), Q6R in CDR-L2 (SEQ ID NO:9),D1G in CDR-H1 (SEQ ID NO:5), Y2H in CDR-H1 (SEQ ID NO:5); A3G in CDR-H1(SEQ ID NO:5), and T3N in CDR-H2 (SEQ ID NO:6). Additional mutationsthat can be incorporated into the improved affinity variant anti-TNF-αantibodies and anti-TNF-α binding fragments can be deimmunizingsubstitutions, such as those described in FIG. 17, as well as othermutations, e.g., substitutions, that do not destroy the ability of theanti-TNF-α antibodies and anti-TNF-α binding fragments to bind TNF-α,including but not limited to the known mutations described in FIGS. 13to 24 or the mutations described in FIG. 31.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments include at least one substitution selected from T4F in CDR-L2,T4W in CDR-L2, T4Y in CDR-L2, L5R in CDR-L2, L5K in CDR-L2, Q6R inCDR-L2, Y2H in CDR-H1, A3G in CDR-H1, and T3N in CDR-H2. Additionalmutations or combinations of mutations that can be incorporated intosuch anti-TNF-α antibodies and anti-TNF-α binding fragments can beselected from one or more of FIGS. 17 and 19 to 31.

In certain other aspects, the anti-TNF-α antibodies and anti-TNF-αbinding fragments include at least one substitution selected from T4F inCDR-L2, T4W in CDR-L2, T4Y in CDR-L2, L5R in CDR-L2, L5K in CDR-L2, Q6Rin CDR-L2, Y2H in CDR-H1, A3G in CDR-H1, and T3N in CDR-H2. Additionalmutations or combinations of mutations that can be incorporated intosuch anti-TNF-α antibodies and anti-TNF-α binding fragments can beselected from one or more of FIGS. 17 and 19 to 24.

In yet other aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments include the substitutions G5S+A11S or G5S+A11G in CDR-L1.Additional mutations or combinations of mutations that can beincorporated into such anti-TNF-α antibodies and anti-TNF-α bindingfragments can be selected from one or more of FIGS. 17-31.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments include the substitutions selected from S3N in CDR-L2, T4V inCDR-L2, Q6K in CDR-L2, and D1G in CDR-H1 in combination with at leastone substitution selected from FIGS. 17, 18 and 31. Additional mutationsor combinations of mutations that can be incorporated into suchanti-TNF-α antibodies and anti-TNF-α binding fragments can be selectedfrom one or more of FIGS. 17 to 30.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments include the substitutions selected from S3N in CDR-L2, T4V inCDR-L2, Q6K in CDR-L2, and D1G in CDR-H1 in combination with at leastone substitution selected from S3K in CDR-L2, S3R in CDR-L2, T4H inCDR-L2, T4Q in CDR-L2, T4F in CDR-L2, T4W in CDR-L2, T4Y in CDR-L2, L5Rin CDR-L2, L5K in CDR-L2, Q6R in CDR-L2, Y2H in CDR-H1, A3G in CDR-H1,and T3N in CDR-H2.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments include the combination of substitutions selected from atleast one of S3K, T4H, L5R and Q6R; S3K, T4Q, L5R and Q6K; S3K, T4Y andL5K; S3K and T4Y; S3N, T4V, L5R and Q6K; S3N, T4W, L5R and Q6R; S3R, T4Fand L5R; S3R, T4F, L5R and Q6R; S3R, T4H and Q6K; S3R, T4W, L5K and Q6R;T4H, L5K and Q6K; T4H, L5K and Q6R; T4W, L5R and Q6R; and T4Y and L5R inCDR-L2, wherein the six CDRs altogether have up to 17 amino acidsubstitutions as compared to CDR sequences of the antibody D2E7. Theanti-TNF-α antibodies or anti-TNF-α binding fragments optionally includeone or more additional mutations or combinations of mutations which canbe selected from one or more of FIGS. 17 to 30.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments include one or more substitutions or combinations ofsubstitutions selected from S3K, S3R, S3N, T4F, T4W, T4Y, T4H, T4Q, T4V,L5R, L5K, Q6R, and Q6K in CDR-L2. Additional mutations or combinationsof mutations that can be incorporated into such anti-TNF-αantibodies andanti-TNF-α binding fragments can be selected from one or more of FIGS.17 to 30.

In other aspects, the present disclosure relates to variants of theanti-TNF-α antibody D2E7 with reduced immunogenicity as compared toD2E7. In certain aspects, the anti-TNF-α antibodies and anti-TNF-αbinding fragments include at least one substitution or combination ofsubstitution(s) in CDR-L1 (SEQ ID NO:8) selected from R7Q; A11S;R7Q+A11S; N8T; N8T+A11S; I6T; A11G; I6T+A11G; Q4G; Q4G+A11S; Q4G+A11G;Q4H; Q4H+A11S; Q4R; Q4R+A11S; G5S; G5S+A11S; N8S+A11S; I6T+A11S; andN8T+A11G. Additional mutations that can be incorporated into theanti-TNF-α antibodies and anti-TNF-α binding fragments with reducedantigenicity include substitutions that improve binding properties toTNF-α, such as those described in FIG. 18 and/or FIG. 31, as well asother mutations, e.g., substitutions that do not destroy the ability ofthe anti-TNF-α antibodies and anti-TNF-α binding fragments to bindTNF-α, including but not limited to the known mutations described inFIGS. 19 to 31.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments of the disclosure have VH and VL sequences having 80% to 99%sequence identity to the VH and VL sequences of D2E7, and include atleast one amino acid substitution in at least one CDR as compared toD2E7. In specific embodiments, the percentage sequence identity for theheavy chain and the light chain compared to the VH and VL sequences ofD2E7 is each independently selected from at least 80%, at least 85%, atleast 90%, or at least 95% sequence identity.

In certain aspects, the anti-TNF-α antibodies and anti-TNF-α bindingfragments of the disclosure have up to 17 amino acid substitutions intheir CDRs as compared to the CDRs of D2E7. Variant antibodies with 17amino acid substitutions that maintain their target binding capabilityhave been generated by Bostrom et al., 2009, Science 323:1610-14. Theanti-TNF-α antibodies and anti-TNF-α binding fragments of the disclosurecan also have up to 16, up to 15, up to 14, up to 13, up to 12, up to11, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, or up to 4amino acid substitutions in their CDRs as compared to CDR sequences ofthe antibody D2E7.

In specific embodiments, an anti-TNF-α antibody or anti-TNF-α bindingfragment of the disclosure has, independently:

-   -   up to one, or up to two, or up to three CDR-H1 substitutions as        compared to the corresponding CDR of D2E7;    -   up to one, up to two, up to three, up to four, up to five or up        to six CDR-H2 substitutions as compared to the corresponding CDR        of D2E7;    -   up to one, up to two, up to three, up to four, or up to five        CDR-H3 substitutions as compared to the corresponding CDR of        D2E7;    -   up to one, up to two, up to three, or up to four CDR-L1        substitutions as compared to the corresponding CDR of D2E7;    -   up to one, up to two, up to three, or up to four CDR-L2        substitutions as compared to the corresponding CDR of D2E7; and    -   up to one, up to two, up to three, or up to four CDR-L3        substitutions as compared to the corresponding CDR of D2E7.

The present disclosure further provides pharmaceutical compositionscomprising modified anti-TNF-α antibodies and anti-TNF-α bindingfragments having increased affinity to TNF-α and/or reducedimmunogenicity as compared to D2E7.

In certain aspects, an anti-TNF-α antibody or anti-TNF-α bindingfragment of the disclosure can be a bispecific antibody or a TNF-αbinding fragment of a bispecific antibody. The bispecific antibody canbe specific to TNF-α and another pro-inflammatory cytokine (such as, forexample, lymphotoxin, interferon-γ, or interleukin-1).

Nucleic acids comprising nucleotide sequences encoding the anti-TNF-αantibodies and anti-TNF-α binding fragments of the disclosure areprovided herein, as are vectors comprising nucleic acids. Additionally,prokaryotic and eukaryotic host cells transformed with a vectorcomprising a nucleotide sequence encoding an anti-TNF-α antibody oranti-TNF-α binding fragment are provided herein, as well as eukaryotic(such as mammalian) host cells engineered to express the nucleotidesequences. Methods of producing anti-TNF-αantibodies and anti-TNF-αbinding fragments by culturing host cells are also provided.

The anti-TNF-α antibodies and anti-TNF-α binding fragments of thedisclosure are useful in the treatment of immune disorders, e.g.,systemic lupus erythematosus, rheumatoid arthritis, thyroidosis, graftversus host disease, scleroderma, diabetes mellitus, Grave's disease,sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, orCrohn's disease.

It should be noted that the indefinite articles “a” and “an” and thedefinite article “the” are used in the present application, as is commonin patent applications, to mean one or more unless the context clearlydictates otherwise. Further, the term “or” is used in the presentapplication, as is common in patent applications, to mean thedisjunctive “or” or the conjunctive “and.”

All publications mentioned in this specification are herein incorporatedby reference. Any discussion of documents, acts, materials, devices,articles or the like that has been included in this specification issolely for the purpose of providing a context for the presentdisclosure. It is not to be taken as an admission that any or all ofthese matters form part of the prior art base or were common generalknowledge in the field relevant to the present disclosure as it existedanywhere before the priority date of this application.

The features and advantages of the disclosure will become furtherapparent from the following detailed description of embodiments thereof.

6. BRIEF DESCRIPTION OF THE TABLES AND FIGURES

FIGS. 1A-1E. FIG. 1A shows the amino acid sequences of the D2E7 heavyand light chains, with CDR regions in bold, underlined text. FIG. 1Bshows the CDR sequences and corresponding sequence identifiers of D2E7.FIG. 1C shows a correspondence chart between the heavy chain CDRnumbering and the heavy chain Kabat numbering. FIG. 1D shows acorrespondence chart between the light chain CDR numbering and the lightchain Kabat numbering. FIG. 1E shows the nucleotide sequences of theheavy and light chain variable regions of D2E7 (SEQ ID NO:1 and SEQ IDNO:3, respectively) as published in U.S. Pat. No. 6,090,382.

FIG. 2 shows percent responses (bottom) and average stimulation indexes(top) to the D2E7 VL peptides.

FIG. 3 shows average stimulation indexes (top) and percent responses(bottom) to the D2E7 VH peptides. Peptide #27 had an anomalousstimulation index in one donor, and is indicated in darker shading.

FIG. 4 shows D2E7 VL CDR1 epitope peptide variants. Open symbolsindicate multiple retests of the unmodified parent peptide within thedataset. Filled symbols represent unique peptide alanine scan variants.The sequence of the most reduced response-inducing variants isindicated.

FIG. 5 shows D2E7 VL CDR1 epitope peptide variants. Open symbolsindicate multiple retests of the unmodified parent peptide within thedataset. Filled symbols represent unique peptide variants. The mostreduced response-inducing variants are indicated by a circle. Thisfigure graphically represents data from FIG. 13.

FIG. 6 shows the results of competition ELISA of D2E7 variantantibodies. ELISA plates were coated with TNF-α. Biotinylated D2E7 wasincluded in all wells at a single concentration, and the variantantibody was titrated in. The IC₅₀ values were calculated for eachantibody. The experiment was performed three times. The Y axis showsaverage results as a percent of the parent antibody binding.

FIG. 7 shows D2E7 VH peptides and D2E7 VL peptides, respectively, thatwere tested for immunogenicity.

FIG. 8 shows identified CD4+ T cell epitope regions in D2E7. CDR regionsare underlined.

FIG. 9 shows HLA class II associations and relative risk of response tothe D2E7 VL region peptide epitopes.

FIG. 10 shows sequences of D2E7 VL DR1 epitope variants, A total of 99donors were tested. The number of responders, the percent of responders,and the average stimulation index is indicated for each peptide tested.

FIG. 11 shows candidate mutations in CDR-L1 for lowering immunogenicityof D2E7. The numbering of the amino acids in FIG. 11 corresponds to thepositions in the context of the D2E7 light chain.

FIG. 12 shows BIAcore and ELISA results for substitutions in CDR-L1 thatdo not result in significantly decreased binding as coipared to D2E7.The numbering of the amino acids in FIG. 12 corresponds to the positionsin the context of the D2E7 lit chain. Improvement in K (as measured byBIAcore) and IC₅₀ of binding (as measured by ELISA) are indicated by“Wix”. CV% refers to the standard deviation as a percentage of the totalvalue measure.

FIG. 13 shows T-cell assay results for all single and double mutationsto the D2E7 epitope. Peptide 1 is the parent peptide. Modifications tothe parent peptide are in bold-faced type.

FIG. 14 shows the preferred epitope peptide variants based solely on Tcell assay results. The numbering of the amino acids in FIG. 14corresponds to the positions in the context of the D2E7 light chain.

FIG. 15 shows anti-proliferation bioactivity of antibodies constructedto contain the preferred variant epitope peptides. The parent isunmodified D2E7 antibody. The numbering of the amino acids in FIG. 15corresponds to the positions in the context of the D2E7 light chain.

FIG. 16 shows binding kinetics of D2E7 and the D2E7 variants againstTNF-α as analyzed by BIAcore. The numbering of the amino acids in FIG.16 corresponds to the positions in the context of the D2E7 light chain.

FIG. 17 shows CDR-L1 substitutions or combinations of substitutions thatcan be incorporated into D2E7-related antibodies to reduce theirimmunogenicity.

FIG. 18 shows CDR amino acid substitutions outside CDR-L1 resulting inimproved K_(D) (as analyzed by BIAcore), affinity (as measured byELISA), or both as compared to D2E7. The numbering of the amino acids inFIG. 18 corresponds to the positions in the context of the D2E7 lightand heavy chains. Improvement in K_(D) (as measured by BIAcore) and IC₅₀of binding (as measured by ELISA) are indicated by “WTx”. CV% refers tothe standard deviation as a percentage of the total value measure and“ND” means “not done”.

FIG. 19 shows known mutations in CDR-H1 that can be incorporated intothe antibodies of the disclosure.

FIG. 20 shows known mutations in CDR-H2 that can be incorporated intothe antibodies of the disclosure. The inclusion of 2 amino acids into asingle cell indicates a CDR variant that incorporates an addition to orinsertion into the CDR. Shading of a cell indicates a CDR variant thatlacks the shaded amino acid residues.

FIG. 21 shows known mutations in CDR-H3 that can be incorporated intothe antibodies of the disclosure.

FIG. 22 shows known mutations in CDR-L1 that can be incorporated intothe antibodies of the disclosure. The inclusion of 2 amino acids into asingle cell indicates a CDR variant that incorporates an addition to orinsertion into the CDR.

FIG. 23 shows known mutations in CDR-L2 that can be incorporated intothe antibodies of the disclosure. The inclusion of 2 amino acids into asingle cell indicates a CDR variant that incorporates the indicatedadditional N-terminal amino acid into the CDR.

FIG. 24 shows known mutations in CDR-L3 that can be incorporated intothe antibodies of the disclosure. The inclusion of 2 amino acids into asingle cell indicates a CDR variant that incorporates the indicatedadditional N-terminal amino acid into the CDR.

FIG. 25 shows further known mutations in CDR-H1 that can be incorporatedinto the antibodies of the disclosure.

FIG. 26 shows further known mutations in CDR-H2 that can be incorporatedinto the antibodies of the disclosure.

FIG. 27 shows further known mutations in CDR-H3 that can be incorporatedinto the antibodies of the disclosure.

FIG. 28 shows further known mutations in CDR-L1 that can be incorporatedinto the antibodies of the disclosure.

FIG. 29 shows further known mutations in CDR-L2 that can be incorporatedinto the antibodies of the disclosure.

FIG. 30 shows further known mutations in CDR-L3 that can be incorporatedinto the antibodies of the disclosure.

FIG. 31 shows combinations of point mutations in CDR-L2 resulting inimproved K_(D) (as analyzed by BIAcore), affinity (as measured byELISA), or both as compared to D2E7. The point mutations can beincorporated singly or in combination into the antibodies of thedisclosure.

7. DETAILED DESCRIPTION 7.1 Anti-TNF-α Antibodies

The present disclosure provides anti-TNF-α antibodies. Unless indicatedotherwise, the term “antibody” (Ab) refers to an immunoglobulin moleculethat specifically binds to, or is immunologically reactive with, aparticular antigen, and includes polyclonal, monoclonal, geneticallyengineered and otherwise modified forms of antibodies, including but notlimited to chimeric antibodies, humanized antibodies, heteroconjugateantibodies (e.g., bispecific antibodies, diabodies, triabodies, andtetrabodies), and antigen binding fragments of antibodies, including,e.g., Fab′, F(ab′)₂, Fab, Fv, rIgG, and scFv fragments. Moreover, unlessotherwise indicated, the term “monoclonal antibody” (mAb) is meant toinclude both intact molecules, as well as, antibody fragments (such as,for example, Fab and F(ab′)₂ fragments) which are capable ofspecifically binding to a protein. Fab and F(ab′)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation ofthe animal or plant, and may have less non-specific tissue binding thanan intact antibody (Wahl et al., 1983, J. Nucl. Med. 24:316).

The term “scFv” refers to a single chain Fv antibody in which thevariable domains of the heavy chain and the light chain from atraditional antibody have been joined to form one chain.

References to “VH” refer to the variable region of an immunoglobulinheavy chain of an antibody, including the heavy chain of an Fv, scFv, orFab. References to “VL” refer to the variable region of animmunoglobulin light chain, including the light chain of an Fv, scFv,dsFv or Fab. Antibodies (Abs) and immunoglobulins (Igs) areglycoproteins having the same structural characteristics. Whileantibodies exhibit binding specificity to a specific target,immunoglobulins include both antibodies and other antibody-likemolecules which lack target specificity. Native antibodies andimmunoglobulins are usually heterotetrameric glycoproteins of about150,000 Daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each heavy chain has at the amino terminus avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at the amino terminus (VL) and aconstant domain at the carboxy terminus.

The anti-TNF-α antibodies of the disclosure bind to human TNF-α andinhibit TNF-αreceptor activity in a cell. Without being bound by any onetheory, the inventors believe that the antibodies reduce the binding ofTNF-α to both the low affinity TNF-α receptor (p75) and the highaffinity TNF-α receptor (p55).

The anti-TNF-α antibodies of the disclosure contain complementaritydetermining regions (CDRs) that are related in sequence to the CDRs ofthe antibody D2E7 (also known as Adalimumab or HUMIRA®).

CDRs are also known as hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework (FR). As is known in the art,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art. Some positions within a variable domainmay be viewed as hybrid hypervariable positions in that these positionscan be deemed to be within a hypervariable region under one set ofcriteria while being deemed to be outside a hypervariable region under adifferent set of criteria. One or more of these positions can also befound in extended hypervariable regions. The disclosure providesantibodies comprising modifications in these hybrid hypervariablepositions. The variable domains of native heavy and light chains eachcomprise four FR regions, largely by adopting a β-sheet configuration,connected by three CDRs, which form loops connecting (and in some casesforming part of) the β-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions in the orderFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and with the CDRs from the other chain,contribute to the formation of the target binding site of antibodies(See Kabat et al., Sequences of Proteins of Immunological Interest(National Institute of Health, Bethesda, Md. 1987)). As used herein,numbering of immunoglobulin amino acid residues is done according to theimmunoglobulin amino acid residue numbering system of Kabat et al.unless otherwise indicated.

The sequences of the heavy and light chain variable regions of D2E7 arerepresented by SEQ ID NO:2 and SEQ ID NO:4, respectively, and encoded bySEQ ID NO.:1 and SEQ ID NO.:3, respectively. The sequences of the heavyand light chain variable regions are also depicted in FIG. 1A. Thesequences of the CDRs of D2E7, and their corresponding identifiers, arepresented in FIG. 1B. The sequences of the heavy and light chainvariable regions of D2E7 (as published in U.S. Pat. No. 6,090,382) areshown in FIG. 1C. Any nucleotide sequences encoding SEQ ID NO:2 or SEQID NO:4 can be used in the compositions and methods of the presentdisclosure.

The present disclosure further provides anti-TNF-α antibody fragmentscomprising CDR sequences that are related to the CDR sequences of D2E7.The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the target binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)2 and Fv fragments. An “Fv”fragment is the minimum antibody fragment which contains a completetarget recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in a tight, noncovalentassociation (VH-VL dimer). It is in this configuration that the threeCDRs of each variable domain interact to define a target binding site onthe surface of the VH-VL dimer. Often, the six CDRs confer targetbinding specificity to the antibody. However, in some instances even asingle variable domain (or half of an Fv comprising only three CDRsspecific for a target) can have the ability to recognize and bindtarget. “Single chain Fv” or “scFv” antibody fragments comprise the VHand VL domains of an antibody in a single polypeptide chain. Generally,the Fv polypeptide further comprises a polypeptide linker between the VHand VL domain that enables the scFv to form the desired structure fortarget binding. “Single domain antibodies” are composed of a single VHor VL domains which exhibit sufficient affinity to the TNF-α. In aspecific embodiment, the single domain antibody is a camelid antibody(see, e.g., Riechmann, 1999, Journal of Immunological Methods231:25-38).

The Fab fragment contains the constant domain of the light chain and thefirst constant domain (CHO of the heavy chain. Fab′ fragments differfrom Fab fragments by the addition of a few residues at the carboxylterminus of the heavy chain CH₁ domain including one or more cysteinesfrom the antibody hinge region. F(ab′) fragments are produced bycleavage of the disulfide bond at the hinge cysteines of the F(ab′)₂pepsin digestion product. Additional chemical couplings of antibodyfragments are known to those of ordinary skill in the art.

In certain embodiments, the anti-TNF-α antibodies of the disclosure aremonoclonal antibodies. The term “monoclonal antibody” as used herein isnot limited to antibodies produced through hybridoma technology. Theterm “monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone andnot the method by which it is produced. Monoclonal antibodies useful inconnection with the present disclosure can be prepared using a widevariety of techniques known in the art including the use of hybridoma,recombinant, and phage display technologies or a combination thereof.The anti-TNF-α antibodies of the disclosure include chimeric,primatized, humanized, or human antibodies.

The anti-TNF-α antibodies of the disclosure can be chimeric antibodies.The term “chimeric” antibody as used herein refers to an antibody havingvariable sequences derived from a non-human immunoglobulin, such as rator mouse antibody, and human immunoglobulin constant regions, typicallychosen from a human immunoglobulin template. Methods for producingchimeric antibodies are known in the art. See, e.g., Morrison, 1985,Science 229(4719):1202-7; Oi et al., 1986, BioTechniques 4:214-221;Gillies et al., 1985, J. Immunol. Methods 125:191-202; U.S. Pat. Nos.5,807,715; 4,816,567; and 4,816,397, which are incorporated herein byreference in their entireties.

The anti-TNF-α antibodies of the disclosure can be humanized.“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other target-binding subdomains of antibodies)which contain minimal sequences derived from non-human immunoglobulin.In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence. The humanized antibody can alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin consensus sequence. Methods ofantibody humanization are known in the art. See, e.g., Riechmann et al.,1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761;5,693,762; and 6,180,370 to Queen et al.; EP239400; PCT publication WO91/09967; U.S. Pat. No. 5,225,539; EP592106; EP519596; Padlan, 1991,Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814;Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat.No. 5,565,332, all of which are hereby incorporated by reference intheir entireties.

The anti-TNF-α antibodies of the disclosure can be human antibodies.Completely “human” anti-TNF-α antibodies can be desirable fortherapeutic treatment of human patients. As used herein, “humanantibodies” include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulin and that do not express endogenous immunoglobulins. Humanantibodies can be made by a variety of methods known in the artincluding phage display methods using antibody libraries derived fromhuman immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO 98/24893;WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741, each of which isincorporated herein by reference in its entirety. Human antibodies canalso be produced using transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins but which can express humanimmunoglobulin genes. See, e.g., PCT publications WO 98/24893; WO92/01047; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Medarex (Princeton,N.J.), Astellas Pharma (Deerfield, Ill.), Amgen (Thousand Oaks, Calif.)and Regeneron (Tarrytown, N.Y.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above. Completely human antibodies that recognize aselected epitope can be generated using a technique referred to as“guided selection.” In this approach a selected non-human monoclonalantibody, e.g., a mouse antibody, is used to guide the selection of acompletely human antibody recognizing the same epitope (Jespers et al.,1988, Biotechnology 12:899-903).

The anti-TNF-α antibodies of the disclosure can be primatized. The term“primatized antibody” refers to an antibody comprising monkey variableregions and human constant regions. Methods for producing primatizedantibodies are known in the art. See e.g., U.S. Pat. Nos. 5,658,570;5,681,722; and 5,693,780, which are incorporated herein by reference intheir entireties.

The anti-TNF-α antibodies of the disclosure can be bispecificantibodies. Bispecific antibodies are monoclonal, often human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present disclosure, one of the bindingspecificities can be directed towards TNF-α, the other can be for anyother antigen, e.g., for a cell-surface protein, receptor, receptorsubunit, tissue-specific antigen, virally derived protein, virallyencoded envelope protein, bacterially derived protein, or bacterialsurface protein, etc.

The anti-TNF-α antibodies of the disclosure include derivatizedantibodies. For example, but not by way of limitation, derivatizedantibodies are typically modified by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein (see Section 7.6 for a discussion of antibodyconjugates), etc. Any of numerous chemical modifications can be carriedout by known techniques, including, but not limited to, specificchemical cleavage, acetylation, formylation, metabolic synthesis oftunicamycin, etc. Additionally, the derivative can contain one or morenon-natural amino acids, e.g., using ambrx technology (See, e.g.,Wolfson, 2006, Chem. Biol. 13(10):1011-2).

In yet another embodiment of the disclosure, the anti-TNF-α antibodiesor fragments thereof can be antibodies or antibody fragments whosesequence has been modified to alter at least one constantregion-mediated biological effector function relative to thecorresponding wild type sequence. For example, in some embodiments, ananti-TNF-αantibody of the disclosure can be modified to reduce at leastone constant region-mediated biological effector function relative to anunmodified antibody, e.g., reduced binding to the Fc receptor (FcγR).FcγR binding can be reduced by mutating the immunoglobulin constantregion segment of the antibody at particular regions necessary for FcγRinteractions (See, e.g., Canfield and Morrison, 1991, J. Exp. Med.173:1483-1491; and Lund et al., 1991, J. Immunol. 147:2657-2662).Reduction in FcγR binding ability of the antibody can also reduce othereffector functions which rely on FcγR interactions, such asopsonization, phagocytosis and antigen-dependent cellular cytotoxicity(“ADCC”).

In other embodiments of the disclosure, an anti-TNF-α antibody orfragment thereof can be modified to acquire or improve at least oneconstant region-mediated biological effector function relative to anunmodified antibody, e.g., to enhance FcγR interactions (See, e.g., US2006/0134709). For example, an anti-TNF-α antibody of the disclosure canhave a constant region that binds FcγRIIA, FcγRIIB and/or FcγRIIIA withgreater affinity than the corresponding wild type constant region.

Thus, antibodies of the disclosure can have alterations in biologicalactivity that result in increased or decreased opsonization,phagocytosis, or ADCC. Such alterations are known in the art. Forexample, modifications in antibodies that reduce ADCC activity aredescribed in U.S. Pat. No. 5,834,597. An exemplary ADCC lowering variantcorresponds to “mutant 3” (shown in FIG. 4 of U.S. Pat. No. 5,834,597)in which residue 236 is deleted and residues 234, 235 and 237 (using EUnumbering) are substituted with alanines.

In some embodiments, the anti-TNF-α antibodies of the disclosure havelow levels of or lack fucose. Antibodies lacking fucose have beencorrelated with enhanced ADCC activity, especially at low doses ofantibody. See Shields et al., 2002, J. Biol. Chem. 277:26733-26740;Shinkawa et al., 2003, J. Biol. Chem. 278:3466-73. Methods of preparingfucose-less antibodies include growth in rat myeloma YB2/0 cells (ATCCCRL 1662). YB2/0 cells express low levels of FUT8 mRNA, which encodesα-1,6-fucosyltransferase, an enzyme necessary for fucosylation ofpolypeptides.

In yet another aspect, the anti-TNF-α antibodies or fragments thereofcan be antibodies or antibody fragments that have been modified toincrease or reduce their binding affinities to the fetal Fc receptor,FcRn, for example, by mutating the immunoglobulin constant regionsegment at particular regions involved in FcRn interactions (See, e.g.,WO 2005/123780). In particular embodiments, an anti-TNF-α antibody ofthe IgG class is mutated such that at least one of amino acid residues250, 314, and 428 of the heavy chain constant region is substitutedalone, or in any combinations thereof, such as at positions 250 and 428,or at positions 250 and 314, or at positions 314 and 428, or atpositions 250, 314, and 428, with positions 250 and 428 a specificcombination. For position 250, the substituting amino acid residue canbe any amino acid residue other than threonine, including, but notlimited to, alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,methionine, asparagine, proline, glutamine, arginine, serine, valine,tryptophan, or tyrosine. For position 314, the substituting amino acidresidue can be any amino acid residue other than leucine, including, butnot limited to, alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, methionine,asparagine, proline, glutamine, arginine, serine, threonine, valine,tryptophan, or tyrosine. For position 428, the substituting amino acidresidues can be any amino acid residue other than methionine, including,but not limited to, alanine, cysteine, aspartic acid, glutamic acid,phenylalanine, glycine, histidine, isoleucine, lysine, leucine,asparagine, proline, glutamine, arginine, serine, threonine, valine,tryptophan, or tyrosine. Specific combinations of suitable amino acidsubstitutions are identified in Table 1 of U.S. Pat. No. 7,217,797,which table is incorporated by reference herein in its entirety. Suchmutations increase the antibody's binding to FcRn, which protects theantibody from degradation and increases its half-life.

In yet other aspects, an anti-TNF-α antibody has one or more amino acidsinserted into one or more of its hypervariable regions, for example asdescribed in Jung and Pliickthun, 1997, Protein Engineering 10:9,959-966; Yazaki et al., 2004, Protein Eng. Des Sel. 17(5):481-9. Epub2004 Aug. 17; and U.S. Pat. App. No. 2007/0280931.

7.2 Nucleic Acids and Expression Systems

The present disclosure encompasses nucleic acid molecules and host cellsencoding the anti-TNF-α antibodies of the disclosure.

An anti-TNF-α antibody of the disclosure can be prepared by recombinantexpression of immunoglobulin light and heavy chain genes in a host cell.To express an antibody recombinantly, a host cell is transfected withone or more recombinant expression vectors carrying DNA fragmentsencoding the immunoglobulin light and heavy chains of the antibody suchthat the light and heavy chains are expressed in the host cell and,optionally, secreted into the medium in which the host cells arecultured, from which medium the antibodies can be recovered. Standardrecombinant DNA methodologies are used to obtain antibody heavy andlight chain genes, incorporate these genes into recombinant expressionvectors and introduce the vectors into host cells, such as thosedescribed in Molecular Cloning; A Laboratory Manual, Second Edition(Sambrook, Fritsch and Maniatis (eds), Cold Spring Harbor, N.Y., 1989),Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds.,Greene Publishing Associates, 1989) and in U.S. Pat. No. 4,816,397.

In one embodiment, the anti-TNF-α antibodies are similar to D2E7 but forchanges in one or more CDRs (referred to hereinbelow as having“D2E7-related” sequences). In another embodiment, the anti-TNF-αantibodies are similar to D2E7 but for changes in one or more frameworkregions. In yet another embodiment, the anti-TNF-α antibodies aresimilar to D2E7 but for changes in one or more CDRs and in one or moreframework regions. To generate nucleic acids encoding such anti-TNF-αantibodies, DNA fragments encoding the light and heavy chain variableregions are first obtained. These DNAs can be obtained by amplificationand modification of germline DNA or cDNA encoding light and heavy chainvariable sequences, for example using the polymerase chain reaction(PCR). Germline DNA sequences for human heavy and light chain variableregion genes are known in the art (See, e.g., the “VBASE” human germlinesequence database; see also Kabat, E. A. et al., 1991, Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242; Tomlinson etal., 1992, J. Mol. Biol. 22T:116-198; and Cox et al., 1994, Eur. J.Immunol. 24:827-836; the contents of each of which are incorporatedherein by reference). A DNA fragment encoding the heavy or light chainvariable region of D2E7, the sequences of which are shown in FIG. 1C,can be synthesized and used as a template for mutagenesis to generate avariant as described herein using routine mutagenesis techniques;alternatively, a DNA fragment encoding the variant can be directlysynthesized.

Once DNA fragments encoding D2E7 or D2E7-related VH and VL segments areobtained, these DNA fragments can be further manipulated by standardrecombinant DNA techniques, for example, to convert the variable regiongenes to full-length antibody chain genes, to Fab fragment genes or to ascFv gene. In these manipulations, a VL- or VH-encoding DNA fragment isoperatively linked to another DNA fragment encoding another protein,such as an antibody constant region or a flexible linker. The term“operatively linked,” as used in this context, is intended to mean thatthe two DNA fragments are joined such that the amino acid sequencesencoded by the two DNA fragments remain in-frame.

The isolated DNA encoding the VH region can be converted to afull-length heavy chain gene by operatively linking the VH-encoding DNAto another DNA molecule encoding heavy chain constant regions (CH₁, CH₂,CH₃ and optionally CH₄). The sequences of human heavy chain constantregion genes are known in the art (See, e.g., Kabat, E. A. et al., 1991,Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242)and DNA fragments encompassing these regions can be obtained by standardPCR amplification. The heavy chain constant region can be an IgG₁, IgG₂,IgG₃, IgG₄, IgA, IgE, IgM or IgD constant region, but in certainembodiments is an IgG₁ or IgG₄ constant region. For a Fab fragment heavychain gene, the VH-encoding DNA can be operatively linked to another DNAmolecule encoding only the heavy chain CH₁ constant region.

The isolated DNA encoding the VL region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the VL-encoding DNA to another DNA molecule encodingthe light chain constant region, CL. The sequences of human light chainconstant region genes are known in the art (See, e.g., Kabat, E. A. etal., 1991, Sequences of Proteins of Immunological Interest, FifthEdition (U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242)) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region, but in certain embodiments isa kappa constant region. To create a scFv gene, the VH- and VL-encodingDNA fragments are operatively linked to another fragment encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄˜Ser)₃,such that the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker (See, e.g., Bird et al., 1988, Science 242:423-426; Huston etal., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al.,1990, Nature 348:552-554).

To express the anti-TNF-α antibodies of the disclosure, DNAs encodingpartial or full-length light and heavy chains, obtained as describedabove, are inserted into expression vectors such that the genes areoperatively linked to transcriptional and translational controlsequences. In this context, the term “operatively linked” is intended tomean that an antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors or, more typically, bothgenes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector by standardmethods (e.g., ligation of complementary restriction sites on theantibody gene fragment and vector, or blunt end ligation if norestriction sites are present). Prior to insertion of the D2E7 orD2E7-related light or heavy chain sequences, the expression vector canalready carry antibody constant region sequences. For example, oneapproach to converting the D2E7 or D2E7-related VH and VL sequences tofull-length antibody genes is to insert them into expression vectorsalready encoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to the CHsegment(s) within the vector and the VL segment is operatively linked tothe CL segment within the vector. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel, GeneExpression Technology: Methods in Enzymology 185 (Academic Press, SanDiego, Calif., 1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Suitable regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, see,e.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al., and U.S. Pat. No. 4,968,615 by Schaffner et al.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure can carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (See, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, puromycin, blasticidin, hygromycin or methotrexate, on a hostcell into which the vector has been introduced. Suitable selectablemarker genes include the dihydrofolate reductase (DHFR) gene (for use inDHFR⁻ host cells with methotrexate selection/amplification) and the neogene (for G418 selection). For expression of the light and heavy chains,the expression vector(s) encoding the heavy and light chains istransfected into a host cell by standard techniques. The various formsof the term “transfection” are intended to encompass a wide variety oftechniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation, lipofection,calcium-phosphate precipitation, DEAE—dextran transfection and the like.

It is possible to express the antibodies of the disclosure in eitherprokaryotic or eukaryotic host cells. In certain embodiments, expressionof antibodies is performed in eukaryotic cells, e.g., mammalian hostcells, for optimal secretion of a properly folded and immunologicallyactive antibody. Exemplary mammalian host cells for expressing therecombinant antibodies of the disclosure include Chinese Hamster Ovary(CHO cells) (including DHFR⁻ CHO cells, described in Urlaub and Chasin,1980, Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFRselectable marker, e.g., as described in Kaufman and Sharp, 1982, Mol.Biol. 159:601-621), NS0 myeloma cells, COS cells, 293 cells and SP2/0cells. When recombinant expression vectors encoding antibody genes areintroduced into mammalian host cells, the antibodies are produced byculturing the host cells for a period of time sufficient to allow forexpression of the antibody in the host cells or secretion of theantibody into the culture medium in which the host cells are grown.Antibodies can be recovered from the culture medium using standardprotein purification methods. Host cells can also be used to produceportions of intact antibodies, such as Fab fragments or scFv molecules.It is understood that variations on the above procedure are within thescope of the present disclosure. For example, it can be desirable totransfect a host cell with DNA encoding either the light chain or theheavy chain (but not both) of an anti-TNF-α antibody of this disclosure.

Recombinant DNA technology can also be used to remove some or all of theDNA encoding either or both of the light and heavy chains that is notnecessary for binding to TNF-α. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of thedisclosure.

In addition, bifunctional antibodies can be produced in which one heavyand one light chain are an antibody of the disclosure and the otherheavy and light chain are specific for an antigen other than TNF-α bycrosslinking an antibody of the disclosure to a second antibody bystandard chemical crosslinking methods. Bifunctional antibodies can alsobe made by expressing a nucleic acid engineered to encode a bifunctionalantibody.

In certain embodiments, dual specific antibodies, i.e. antibodies thatbind TNF-α and an unrelated antigen using the same binding site, can beproduced by mutating amino acid residues in the light chain and/or heavychain CDRs. In various embodiments, dual specific antibodies that bindTNF-α and another antigen, for example, another proinflammatory cytokine(such as, for example, lymphotoxin, interferon-γ, or interleukin-1) canbe produced by mutating amino acid residues in the periphery of theantigen binding site (See, e.g., Bostrom et al., 2009, Science323:1610-1614). Dual functional antibodies can be made by expressing anucleic acid engineered to encode a dual specific antibody.

For recombinant expression of an anti-TNF-α antibody of the disclosure,the host cell can be co-transfected with two expression vectors of thedisclosure, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide.Typically, the two vectors each contain a separate selectable marker.Alternatively, a single vector can be used which encodes both heavy andlight chain polypeptides.

Once a nucleic acid encoding one or more portions of D2E7 or of ananti-TNF-α antibody with CDR sequences related to the CDR sequences ofD2E7 is generated, further alterations or mutations can be introducedinto the coding sequence, for example to generate nucleic acids encodingantibodies with different CDR sequences, antibodies with reducedaffinity to the Fc receptor, or antibodies of different subclasses.

The anti-TNF-α antibodies of the disclosure can also be produced bychemical synthesis (e.g., by the methods described in Solid PhasePeptide Synthesis, 2^(nd) ed., 1984 The Pierce Chemical Co., Rockford,Ill.). Variant antibodies can also be generated using a cell-freeplatform (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche MolecularBiologicals)).

Once an anti-TNF-α antibody of the disclosure has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor TNF-α after Protein A or Protein G selection, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theanti-TNF-α antibodies of the present disclosure or fragments thereof canbe fused to heterologous polypeptide sequences described herein orotherwise known in the art to facilitate purification.

Once isolated, an anti-TNF-α antibody can, if desired, be furtherpurified, e.g., by high performance liquid chromatography (See, e.g.,Fisher, Laboratory Techniques In Biochemistry And Molecular Biology(Work and Burdon, eds., Elsevier, 1980)), or by gel filtrationchromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala,Sweden).

7.3 Biological Activities of Anti-TNF-α Antibodies

In certain embodiments, the anti-Tnf-α antibodies of the disclosure havecertain biological activities, such as competing with D2E7 for bindingto TNF-α or neutralizing TNF-α activity.

Accordingly, in certain embodiments, anti-TNF-α antibodies of thedisclosure compete with D2E7 for binding to TNF-α. The ability tocompete for binding to TNF-α can be tested using a competition assay. Inone example of a competition assay, TNF-α is adhered onto a solidsurface, e.g., a microwell plate, by contacting the plate with asolution of TNF-α (e.g., at a concentration of 1 μg/mL in PBS over nightat 4° C.). The plate is washed (e.g., 0.1% Tween 20 in PBS) and blocked(e.g., in Superblock, Thermo Scientific, Rockford, Ill.). A mixture ofsub-saturating amount of biotinylated D2E7 (80 ng/mL) and unlabeled D2E7(the “reference” antibody) or competing anti-TNF-α antibody (the “test”antibody) antibody in serial dilution (e.g., at a concentration of 2.8μg/mL, 8.3 μg/mL, or 25 μg/mL) in ELISA buffer (e.g., 1% BSA and 0.1%Tween 20 in PBS) is added to wells and plates are incubated for 1 hourwith gentle shaking. The plate is washed, 1 μg/mL HRP-conjugatedStreptavidin diluted in ELISA buffer was added to each well and theplates incubated for 1 hour. Plates are washed and bound antibodies weredetected by addition of substrate (e.g., TMB, Biofx Laboratories Inc.,Owings Mills, Md.). The reaction is terminated by addition of stopbuffer (e.g., Bio FX Stop Reagents, Biofx Laboratories Inc., OwingsMills, Md.) and the absorbance was measured at 650 nm using microplatereader (e.g., VERSAmax, Molecular Devices, Sunnyvale, Calif.).Variations on this competition assay can also be used to testcompetition between an anti-TNF-α antibody of the disclosure and D2E7.For example, in certain aspects, the anti-TNF-α antibody is used as areference antibody and D2E7 is used as a test antibody. Additionally,instead of soluble TNF-α, membrane-bound TNF-α expressed on cell surface(for example mammalian cells such as 293S) in culture can be used.Alternatively, instead of soluble D2E7 and test antibodies, thoseexpressed on cell surface (for example mammalian cells such as 293c18)in culture can be used too. Generally, about 10⁴ to 10⁶ transfectants,e.g., about 10⁵ transfectants, are used. Other formats for competitionassays are known in the art and can be employed.

In various embodiments, an anti-TNF-α antibody of the disclosure reducesthe binding of labeled D2E7 by at least 40%, by at least 50%, by atleast 60%, by at least 70%, by at least 80%, by at least 90%, or by apercentage ranging between any of the foregoing values (e.g., ananti-TNF-α antibody of the disclosure reduces the binding of labeledD2E7 by 50% to 70%) when the anti-TNF-α antibody is used at aconcentration of 0.08 μg/mL, 0.4 μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL, 100μg/mL or at a concentration ranging between any of the foregoing values(e.g., at a concentration ranging from 2 μg/mL to 10 μg/mL).

In other embodiments, D2E7 reduces the binding of a labeled anti-TNF-αantibody of the disclosure by at least 40%, by at least 50%, by at least60%, by at least 70%, by at least 80%, by at least 90%, or by apercentage ranging between any of the foregoing values (e.g., D2E7reduces the binding of a labeled an anti-TNF-α antibody of thedisclosure by 50% to 70%) when D2E7 is used at a concentration of 0.4μg/mL, 2 μg/mL, 10 μg/mL, 50 μg/mL, 250 μg/mL or at a concentrationranging between any of the foregoing values (e.g., at a concentrationranging from 2 μg/mL to 10 μg/mL).

In other aspects, an anti-TNF-α antibody of the disclosure inhibitsTNF-α activity in a range of in vitro assays, such as cell cytotoxicity,mitogenesis, cytokine induction, and induction of adhesion molecules.Alternatively, activity of an anti-TNF-α antibody of the disclosure canbe measured by in vitro assays using membrane bound TNF-α naturally orrecombinantly expressed on cells, such as ability to induce reversesignaling, cytokine induction, induction of adhesion molecules, CDC andADCC. An exemplary TNF-αneutralization assay that measures inhibition ofsoluble TNF-α cytotoxicity using cells sensitive to TNF-α (e.g., L929)is described below. Other TNF-α cytotoxicity assays can also be used toassess the activity of the anti-TNF-α antibodies of the disclosure.

Thus, in an exemplary embodiment, an anti-TNF-α cytotoxicity assaysentails plating 3×10⁴ murine L929 cells into individual wells of a flatbottomed 96-well microtiter plate. The cells are incubated overnight at37° C. in a humidified 5% CO₂ incubator. The next day, serial dilutionsof the anti-TNF-α antibody (e.g., 0.712 μg/mL, 0.949 μg/mL, 1.27 μg/mL,1.69 μg/mL, 2.25 μg/mL or 3 μg/mL) are prepared in 25 μL of serum-freemedium and added to cells (e.g. final concentration in 150 μL culture is119 ng/mL, 158 ng/mL, 211 ng/mL, 282 ng/mL, 375 ng/mL or 500 ng/mL).After a 2-hour incubation at 37° C., 5% CO₂, TNF-α is added at finalconcentration of 40 ng/mL (e.g., 25 μL of 240 ng/mL) and the cells werefurther incubated for 48 hours at 37° C., 5% CO₂. The wells are scoredfor cytotoxicity as compared to control plates (which in certainembodiments were treated with TNF-α that were not incubated with ananti-TNF-α antibody, e.g., were incubated with an isotype controlantibody and in other embodiments were treated with D2E7) using aviability assay (e.g., CellTiter-Blue, Promega, Madison, Wis.). Otherformats for TNF-α neutralization assays are known in the art and can beemployed.

In various embodiments, an anti-TNF-α antibody of the disclosureneutralizes TNF-α by at least 30%, by at least 40%, by at least 50%, byat least 60%, by at least 70%, by at least 80%, by at least 90%, or by apercentage ranging between any of the foregoing values (e.g., ananti-TNF-α antibody of the disclosure neutralizes TNF-α activity by 50%to 70%) when the anti-TNF-α antibody is used at a concentration of 2ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 0.1 μg/mL, 0.2 μg/mL, 1 μg/mL, 2μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, or at a concentration rangingbetween any of the foregoing values (e.g., at a concentration rangingfrom 1 μg/mL to 5 μg/mL). In some embodiments, an anti-TNF-α antibody ofthe disclosure is at least 80% as effective, at least 90% as effective,at least 100% as effective, at least 110% as effective, at least 125% aseffective or at least 150% as effective, and up to 110% as effective, upto 125% as effective, up to 150% as effective or up to 200% as effectiveas D2E7 at neutralizing TNF-α, or any range between any pair of theforegoing values (e.g., 80% to 125% as effective as D2E7 or 125% to 200%as effective as D2E7 in neutralizing TNF-α).

In certain embodiments, the anti-TNF-α antibodies of the disclosure havea high binding affinity for TNF-α. In specific embodiments, theanti-TNF-α antibodies of the present disclosure have specificassociation rate constants (k_(on) or k_(a) values), dissociation rateconstants (k_(off) or k_(d) values), affinity constants (K_(A) values),dissociation constants (K_(D) values) and/or IC₅₀ values. In certainaspects, such values are selected from the following embodiments.

7.4 Kinetic Properties of Anti-TNF-α Antibodies

In a specific embodiment, an anti-TNF-α antibody of the disclosure bindsto TNF-α with a k_(on) of at least 10⁵ M⁻¹s⁻¹, at least 5×10⁵ M⁻¹s⁻¹, atleast 10⁶ M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least 10⁷ M⁻¹s⁻¹, at least5×10⁷ M⁻¹s⁻¹, at least 10⁸ M⁻¹s⁻¹, or with a k_(on) of any range betweenany pair of the foregoing values (e.g., 5×10⁵ to 5×10⁶ M⁻¹s⁻¹ or 10⁷ to10⁸ M⁻¹s⁻¹).

In another embodiment, an anti-TNF-α antibody of the disclosure binds toTNF-α with a k_(off) rate of 5×10⁻¹ s⁻¹ or less, 10⁻¹ s⁻¹ or less,5×10⁻² s⁻¹ or less, 10⁻² s⁻¹ or less, 5×10⁻³ s⁻¹ or less, 10⁻³ s⁻¹ orless, 5×10⁻⁴ s⁻¹ or less, 10⁻⁴s⁻¹ or less, 5×10⁻⁵s⁻¹ or less, 10⁻⁵s⁻¹ orless, 5×10⁻⁶ s⁻¹ or less, 10⁻⁶ s⁻¹ or less, 5×10⁻⁷ s⁻¹ or less, 10⁻⁷ s⁻¹or less, 5×10⁻⁸ s⁻¹ or less, 10⁻⁸ s⁻¹ or less, 5×10⁻⁹ s⁻¹ or less, 10⁻⁹s⁻¹ or less, 5×10⁻¹⁰ s⁻¹ or less, 10⁻¹⁰ s⁻¹ or less, or with a k_(off)rate of any range between any pair of the foregoing values (e.g., 5×10⁻⁴to 10⁻⁶ s⁻¹, or 5×10⁻⁵ to 5×10⁻⁸ s⁻¹).

In another embodiment, an anti-TNF-α antibody of the disclosure binds toTNF-α with a K_(A) (k_(on)/k_(off)) of at least 10¹¹ nM⁻¹, at least5×10¹¹ nM⁻¹, at least 10¹² nM⁻¹, at least 5×10¹² nM⁻¹, at least 10¹³nM⁻¹, at least 5×10¹³ nM⁻¹, at least 10¹⁴ nM⁻¹, at least 5×10¹⁴ nM⁻¹, atleast 10¹⁵ nM⁻¹, at least 5×10¹⁵ nM⁻¹, at least 10¹⁶ nM⁻¹, at least5×10¹⁶ nM⁻¹, at least 10¹⁷ nM⁻¹, at least 5×10¹⁷ nM⁻¹, at least 10¹⁸nM⁻¹, at least 5×10¹⁸ nM⁻¹, at least 10¹⁹ nM⁻¹, at least 5×10¹⁹ nM⁻¹, atleast 10²⁰ nM⁻¹, at least 5×10²⁰ nM⁻¹, at least 10²¹ nM⁻¹, at least5×10²¹ nM⁻¹, at least 10²² nM⁻¹, at least 5×10²² nM⁻¹, at least 10²³nM⁻¹, at least 5×10²³ nM⁻¹, at least 10²⁴ nM⁻¹, at least 5×10²⁴ nM⁻¹, orwith a K_(A) of any range between any pair of the foregoing values(e.g., 5×10¹⁴ to 10²² nM⁻¹, or 10¹¹ to 5×10¹⁸ nM⁻¹).

In other embodiments, an anti-TNF-α antibody of the disclosure binds toTNF-α with a K_(D) (k_(off)/k_(on)) of 5×10⁷ nM or less, 10⁷ nM or less,5×10⁶ nM or less, 10⁶ nM or less, 5×10⁵ nM or less, 10⁵ nM or less,5×10⁴ nM or less, 10⁴ nM or less, 5×10³ nM or less, 10³ nM or less,5×10² nM or less, 100 nM or less, 90 nM or less, 80 nM or less, 70 nM orless, 60 nM or less, 50 nM or less, 20 nM or less, 15 nM or less, 10 nMor less, 5 nM or less, 3.8 nM or less, 2 nM or less, 1.5 nM or less, 1nM or less, 5×10⁻¹ nM or less, 10⁻¹ nM or less, 5×10⁻² nM or less, 10⁻²nM or less, 5×10⁻³ nM or less, 10⁻³ nM or less, 5×10⁻⁴ nM or less, 10⁻⁴nM or less, 5×10⁻⁵ nM or less, 10⁻⁵ nM or less, 5×10⁻⁶ nM or less, 10⁻⁶nM or less, or with a K_(D) of any range between any pair of theforegoing values (e.g., 5×10⁷ to 50 nM, or 15 nM to 5×10⁻³ nM).

In certain specific embodiments, an TNF-α antibody of the disclosurebinds to TNF-α with a K_(D) (k_(off)/k_(on)) between approximately 0.1nM and approximately 1 nM, or approximately 0.1 nM and approximately 2nM, or approximately 0.1 nM and approximately 3 nM, or approximately 0.1nM and approximately 4 nM, or approximately 0.1 nM and approximately 5nM, or approximately 0.1 nM and approximately 6 nM, or approximately 0.1nM and approximately 7 nM, or approximately 0.1 nM and approximately 8nM, or approximately 0.1 nM and approximately 9 nM, or approximately 0.1nM and approximately 10 nM, or between approximately 0.01 nM andapproximately 0.1 nM, or between approximately 0.01 nM and approximately1 nM, or between approximately 0.01 nM and approximately 2 nM, orbetween approximately 0.01 nM and approximately 3 nM, or betweenapproximately 0.01 nM and approximately 4 nM, or between approximately0.01 nM and approximately 5 nM, or between approximately 0.01 nM andapproximately 6 nM, or between approximately 0.01 nM and approximately 7nM, or between approximately 0.01 nM and approximately 8 nM, or betweenapproximately 0.01 nM and approximately 9 nM, or between approximately0.6 nM and approximately 1.1 nM, or between approximately 0.7 nM andapproximately 1.2 nM, or between approximately 0.5 and approximately 5nM. In other specific embodiments, an anti-TNF-α antibody binds to TNF-αwith a K_(D) (k_(off)/k_(on)) of about 5 nM, about 3.5 nM, about 1.5 nM,about 1 nM, about 0.5 nM, about 0.1 nM, about 0.05 nM or about 0.01 nM.In specific embodiments, the K_(D) (k_(off)/k_(on)) value is determinedby assays well known in the art or described herein, e.g., ELISA,isothermal titration calorimetry (ITC), BIAcore, or fluorescentpolarization assay.

In some embodiments, an anti-TNF-α antibody of the disclosure binds toTNF-α and inhibits the binding of TNF-α to p55, p75 or both at an IC₅₀value of less than 5×10⁷ nM, less than 10⁷ nM, less than 5×10⁶ nM, lessthan 10⁶ nM, less than 5×10⁵ nM, less than 10⁵ nM, less than 5×10⁴ nM,less than 10⁴ nM, less than 5×10³ nM, less than 10³ nM, less than 5×10²nM, less than 100 nM, less than 90 nM, less than 80 nM, less than 70 nM,65 nM, less than 60 nM, less than 50 nM, less than 40 nM, less than 30nM, less than 25 nM, less than 20 nM, less than 15 nM, less than 12 nM,less than 10 nM, less than 5 nM, less than 1 nM, less than 5×10⁻¹ nM,less than 10⁻¹ nM, less than 5×10⁻² nM, less than 10⁻² nM, less than5×10⁻³ nM, less than 10⁻³ nM, less than 5×10⁻⁴ nM, or less than 10⁻⁴ nM,or with an IC₅₀ of any range between any pair of the foregoing values(e.g., 5×10⁷ to 50 nM, or 15 nM to 5×10⁻³ nM). IC₅₀ can be measuredaccording to methods well known in the art or described herein, e.g.,ELISA.

In other embodiments, an anti-TNF-α antibody of the disclosure binds toTNF-α and neutralizes TNF-α at an IC₅₀ value of less than 5×10⁷ nM, lessthan 10⁷ nM, less than 5×10⁶ nM, less than 10⁶ nM, less than 5×10⁵ nM,less than 10⁵ nM, less than 5×10⁴ nM, less than 10⁴ nM, less than 5×10³nM, less than 10³ nM, less than 5×10² nM, less than 100 nM, less than 90nM, less than 80 nM, less than 70 nM, 65 nM, less than 60 nM, less than50 nM, less than 40 nM, less than 30 nM, less than 25 nM, less than 20nM, less than 15 nM, less than 12 nM, less than 10 nM, less than 5 nM,less than 1 nM, less than 5×10⁻¹ nM, less than 10⁻¹ nM, less than 5×10⁻²nM, less than 10⁻² nM, less than 5×10⁻³ nM, less than 10⁻³ nM, less than5×10⁻⁴ nM, or less than 10⁻⁴ nM, or with an IC₅₀ of any range betweenany pair of the foregoing values (e.g., 5×10⁷ to 50 nM, or 15 nM to5×10⁻³ nM). An exemplary neutralization assay that can be used tomeasure the IC₅₀ of an anti-TNF-α antibody is described in Section 7.5below.

In certain specific embodiments, an anti-TNF-α antibody binds to TNF-αand inhibits the binding of TNF-α to p55, p75 or both, or inhibits TNF-αactivity in a TNF-αneutralization assay, at an IC₅₀ value of betweenapproximately 1 nM and approximately 10 nM, between approximately 1 nMand approximately 15 nM, between approximately 1 nM and approximately 20nM, between approximately 1 nM and approximately 25 nM, betweenapproximately 1 nM and approximately 30 nM, between approximately 1 nMand approximately 40 nM, between approximately 1 nM and approximately 50nM, between approximately 10 nM and approximately 10² nM, betweenapproximately 10² nM and approximately 10³ nM, between approximately 10nM and approximately 10⁴ nM, between approximately 10⁴ nM andapproximately 10⁵ nM, between approximately 10⁵ nM and approximately 10⁶nM, or between approximately 10⁶ nM and approximately 10⁷ nM.

In other specific embodiments, an anti-TNF-α antibody binds to TNF-α andinhibits the binding of TNF-α to p55, p75 or both, or inhibits TNF-αactivity in a TNF-α neutralization assay, at an IC₅₀ value of betweenapproximately 5 nM and approximately 10 nM, between approximately 5 nMand approximately 15 nM, between approximately 10 nM and approximately15 nM, between approximately 10 nM and approximately 20 nM, betweenapproximately 10 nM and approximately 30 nM, between approximately 10 nMand approximately 40 nM, between approximately 10 nM and approximately50 nM, between approximately 1 nM and approximately 100 nM, betweenapproximately 10 nM and approximately 100 nM, between approximately 20nM and approximately 100 nM, between approximately 30 nM andapproximately 100 nM, between approximately 40 nM and approximately 100nM, between approximately 50 nM and approximately 100 nM, betweenapproximately 15 nM and approximately 25 nM, or between approximately 15nM and approximately 20 nM.

In certain aspects of the foregoing embodiments, the IC₅₀ is measured inthe presence of TNF-α at a concentration of 0.001 μM, 0.005 μM, 0.01 μM,0.05 μM, 0.1 μM, 0.5 μM, 1 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM,70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700μM, 800 μM, 900 μM, 1000 μm or at a concentration of any range betweenany pair of the foregoing values (e.g., 0.01 to 50 μM, or 10 μM to 100μM).

In certain embodiments, the kinetic properties of an antibody of thedisclosure are comparable to, or improved relative to, the D2E7 antibodyin a comparable assay. For example, in certain embodiments, ananti-TNF-α antibody of the disclosure binds to TNF-α with a k_(on) rateranging from 0.2× to 5× of the k_(on) of D2E7, for example a k_(on) of0.2× of the k_(on) of D2E7, a k_(on) of 0.3× of the k_(on) of D2E7, ak_(on) of 0.4× of the k_(on) of D2E7, a k_(on) of 0.5× of the k_(on) ofD2E7, a k_(on) of 0.6× of the k_(on) of D2E7, a k_(on) of 0.7× of thek_(on) of D2E7, a k_(on) of 0.8× of the k_(on) of D2E7, a k_(on) of 0.9×of the k_(on) of D2E7, a k_(on) of 1× of the k_(on) of D2E7, a k_(on) of1.1× of the k_(on) of D2E7, a k_(on) of 1.2× of the k_(on) of D2E7, ak_(on) of 1.3× of the k_(on) of D2E7, a k_(on) of 1.4× of the k_(on) ofD2E7, a k_(on) of 1.5× of the k_(on) of D2E7, a k_(on) of 1.75× of thek_(on) of D2E7, a k_(on) of 2× of the k_(on) of D2E7, a k_(on) of 2.25×of the k_(on) of D2E7, a k_(on) of 2.5× of the k_(on) of D2E7, a k_(on)of 2.75× of the k_(on) of D2E7, a k_(on) of 3× of the k_(on) of D2E7, ak_(on) of 3.5× of the k_(on) of D2E7, a k_(on) of 4× of the k_(on) ofD2E7, a k_(on) of 4.5× of the k_(on) of D2E7, a k_(on) of 5× of thek_(on) of D2E7, or a k_(on) ranging between any pair of the foregoingvalues, e.g., a k_(on) of 0.7×-1.5× of the k_(on) of D2E7, a k_(on) of0.9×-1.3× of the k_(on) of D2E7, a k_(on) of 0.8×-2× of the k_(on) ofD2E7, a k_(on) of 0.9×-3× of the k_(on) of D2E7, etc.

In embodiments, an anti-TNF-α antibody of the disclosure binds to TNF-αwith a k_(off) rate ranging from 0.2× to 5× of the k_(off) of D2E7, forexample a k_(off) of 0.2× of the k_(off) of D2E7, a k_(off) of 0.3× ofthe k_(off) of D2E7, a k_(off) of 0.4× of the k_(off) of D2E7, a k_(off)of 0.5× of the k_(off) of D2E7, a k_(off) of 0.6× of the k_(off) ofD2E7, a k_(off) of 0.7× of the k_(off) of D2E7, a k_(off) of 0.8× of thek_(off) of D2E7, a k_(off) of 0.9× of the k_(off) of D2E7, a k_(off) of1× of the k_(off) of D2E7, a k_(off) of 1.1× of the k_(off) of D2E7, ak_(off) of 1.2× of the k_(off) of D2E7, a k_(off) of 1.3× of the k_(off)of D2E7, a k_(off) of 1.4× of the k_(off) of D2E7, a k_(off) of 1.5× ofthe k_(off) of D2E7, a k_(off) of 1.75× of the k_(off) of D2E7, ak_(off) of 2× of the k_(off) of D2E7, a k_(off) of 2.25× of the k_(off)of D2E7, a k_(off) of 2.5× of the k_(off) of D2E7, a k_(off) of 2.75× ofthe k_(off) of D2E7, a k_(off) of 3× of the k_(off) of D2E7, a k_(off)of 3.5× of the k_(off) of D2E7, a k_(off) of 4× of the k_(off) of D2E7,a k_(off) of 4.5× of the k_(off) of D2E7, a k_(off) of 5× of the k_(off)of D2E7, or a k_(off) ranging between any pair of the foregoing values,e.g., a k_(off) of 0.7×-1.5× of the k_(off) of D2E7, a k_(off) of0.9×-1.3× of the k_(off) of D2E7, a k_(off) of 0.8×-2× of the k_(off) ofD2E7, a k_(off) of 0.9×-3× of the k_(off) of D2E7, etc.

In other embodiments, an anti-TNF-α antibody of the disclosure binds toTNF-α with a K_(A) (k_(on)/k_(off)) ranging from 0.04× to 25× of theK_(A) of D2E7, for example a K_(A) of 0.04× of the K_(A) of D2E7, aK_(A) of 0.1× of the K_(A) of D2E7, a K_(A) of 0.25× of the K_(A) ofD2E7, a K_(A) of 0.5× of the K_(A) of D2E7, a K_(A) of 0.6× of the K_(A)of D2E7, a K_(A) of 0.7× of the K_(A) of D2E7, a K_(A) of 0.8× of theK_(A) of D2E7, a K_(A) of 0.9× of the K_(A) of D2E7, a K_(A) of 1× ofthe K_(A) of D2E7, a K_(A) of 1.1× of the K_(A) of D2E7, a K_(A) of1.25× of the K_(A) of D2E7, a K_(A) of 1.5× of the K_(A) of D2E7, aK_(A) of 1.75× of the K_(A) of D2E7, a K_(A) of 2× of the K_(A) of D2E7,a K_(A) of 2.5× of the K_(A) of D2E7, a K_(A) of 3× of the K_(A) ofD2E7, a K_(A) of 4× of the K_(A) of D2E7, a K_(A) of 4×% of the K_(A) ofD2E7, a K_(A) of 5× of the K_(A) of D2E7, a K_(A) of 7.5× of the K_(A)of D2E7, a K_(A) of 10× of the K_(A) of D2E7, a K_(A) of 12.5× of theK_(A) of D2E7, a K_(A) of 15× of the K_(A) of D2E7, a K_(A) of 20× ofthe K_(A) of D2E7, a K_(A) of 25× of the K_(A) of D2E7, or a K_(A)ranging between any pair of the foregoing values, e.g., a K_(A) of0.7×-1.25× of the K_(A) of D2E7, a K_(A) of 0.9×-1.5× of the K_(A) ofD2E7, a K_(A) of 0.9×-2× of the K_(A) of D2E7, a K_(A) of 0.8×-1.75× ofthe K_(A) of D2E7, a K_(A) of 0.9×-5× of the K_(A) of D2E7, or any valueor range that can be calculated from the k_(on) and k_(off) ratesdisclosed herein.

In other embodiments, an anti-TNF-α antibody of the disclosure binds toTNF-α a K_(D) (k_(off)/k_(on)) ranging from ranging from 0.04× to 25× ofthe K_(D) of D2E7, for example a K_(D) of 0.04× of the K_(D) of D2E7, aK_(D) of 0.1× of the K_(D) of D2E7, a K_(D) of 0.25× of the K_(D) ofD2E7, a K_(D) of 0.5× of the K_(D) of D2E7, a K_(D) of 0.6× of the K_(D)of D2E7, a K_(D) of 0.7× of the K_(D) of D2E7, a K_(D) of 0.8× of theK_(D) of D2E7, a K_(D) of 0.9× of the K_(D) of D2E7, a K_(D) of 1× ofthe K_(D) of D2E7, a K_(D) of 1.1× of the K_(D) of D2E7, a K_(D) of1.25× of the K_(D) of D2E7, a K_(D) of 1.5× of the K_(D) of D2E7, aK_(D) of 1.75× of the K_(D) of D2E7, a K_(D) of 2× of the K_(D) of D2E7,a K_(D) of 2.5× of the K_(D) of D2E7, a K_(D) of 3× of the K_(D) ofD2E7, a K_(D) of 4× of the K_(D) of D2E7, a K_(D) of 4×% of the K_(D) ofD2E7, a K_(D) of 5× of the K_(D) of D2E7, a K_(D) of 7.5× of the K_(D)of D2E7, a K_(D) of 10× of the K_(D) of D2E7, a K_(D) of 12.5× of theK_(D) of D2E7, a K_(D) of 15× of the K_(D) of D2E7, a K_(D) of 20× ofthe K_(D) of D2E7, a K_(D) of 25× of the K_(D) of D2E7, or a K_(D)ranging between any pair of the foregoing values, e.g., a K_(D) of0.7×-1.25× of the K_(D) of D2E7, a K_(D) of 0.9×-1.5× of the K_(D) ofD2E7, a K_(D) of 0.9×-2× of the K_(D) of D2E7, a K_(D) of 0.8×-1.75× ofthe K_(D) of D2E7, a K_(D) of 0.9×-5× of the K_(D) of D2E7, or any valueor range that can be calculated from the k_(on) and k_(off) ratesdisclosed herein.

In some embodiments, an anti-TNF-α antibody of the disclosure binds toTNF-α and inhibits the binding of TNF-α to p55, p75 or both at an IC₅₀value ranging from 50% to 200% of the IC₅₀ of D2E7, for example an IC₅₀of 50% of the IC₅₀ of D2E7, an IC₅₀ of 60% of the IC₅₀ of D2E7, an IC₅₀of 70% of the IC₅₀ of D2E7, an IC₅₀ of 75% of the IC₅₀ of D2E7, an IC₅₀of 80% of the IC₅₀ of D2E7, an IC₅₀ of 90% of the IC₅₀ of D2E7, an IC₅₀of 95% of the IC₅₀ of D2E7, an IC₅₀ of 100% of the IC₅₀ of D2E7, an IC₅₀of 110% of the IC₅₀ of D2E7, an IC₅₀ of 120% of the IC₅₀ of D2E7, anIC₅₀ of 125% of the IC₅₀ of D2E7, an IC₅₀ of 130% of the IC₅₀ of D2E7,an IC₅₀ of 140% of the IC₅₀ of D2E7, an IC₅₀ of 150% of the IC₅₀ ofD2E7, an IC₅₀ of 160% of the IC₅₀ of D2E7, an IC₅₀ of 170% of the IC₅₀of D2E7, an IC₅₀ of 175% of the IC₅₀ of D2E7, an IC₅₀ of 180% of theIC₅₀ of D2E7, an IC₅₀ of 190% of the IC₅₀ of D2E7, an IC₅₀ of 200% ofthe IC₅₀ of D2E7, or an IC₅₀ of any range between any pair of theforegoing values, e.g., an IC₅₀ of 75%-125% of the IC₅₀ of D2E7, an IC₅₀of 90%-130% of the IC₅₀ of D2E7, an IC₅₀ of 95%-125% of the IC₅₀ ofD2E7, an IC₅₀ of 90%-110% of the IC₅₀ of D2E7, an IC₅₀ of 90%-180% ofthe IC₅₀ of D2E7, or an IC₅₀ of 80%-175% of the IC₅₀ of D2E7. In otherembodiments, a single CDR substitution can result in the foregoingdifferences in IC₅₀ as compared to D2E7, whereas an anti-TNF-α antibodyof the disclosure can comprise such substitution and up to 16 additionalsubstitutions as compared to D2E7.

In other embodiments, an anti-TNF-α antibody of the disclosure binds toTNF-α and neutralizes TNF-α at an IC₅₀ value ranging from 50% to 200% ofthe IC₅₀ of D2E7, for example an IC₅₀ of 50% of the IC₅₀ of D2E7, anIC₅₀ of 60% of the IC₅₀ of D2E7, an IC₅₀ of 70% of the IC₅₀ of D2E7, anIC₅₀ of 75% of the IC₅₀ of D2E7, an IC₅₀ of 80% of the IC₅₀ of D2E7, anIC₅₀ of 90% of the IC₅₀ of D2E7, an IC₅₀ of 95% of the IC₅₀ of D2E7, anIC₅₀ of 100% of the IC₅₀ of D2E7, an IC₅₀ of 110% of the IC₅₀ of D2E7,an IC₅₀ of 120% of the IC₅₀ of D2E7, an IC₅₀ of 125% of the IC₅₀ ofD2E7, an IC₅₀ of 130% of the IC₅₀ of D2E7, an IC₅₀ of 140% of the IC₅₀of D2E7, an IC₅₀ of 150% of the IC₅₀ of D2E7, an IC₅₀ of 160% of theIC₅₀ of D2E7, an IC₅₀ of 170% of the IC₅₀ of D2E7, an IC₅₀ of 175% ofthe IC₅₀ of D2E7, an IC₅₀ of 180% of the IC₅₀ of D2E7, an IC₅₀ of 190%of the IC₅₀ of D2E7, an IC₅₀ of 200% of the IC₅₀ of D2E7, or an IC₅₀ ofany range between any pair of the foregoing values, e.g., an IC₅₀ of75%-125% of the IC₅₀ of D2E7, an IC₅₀ of 90%-130% of the IC₅₀ of D2E7,an IC₅₀ of 95%-125% of the IC₅₀ of D2E7, an IC₅₀ of 90%-110% of the IC₅₀of D2E7, an IC₅₀ of 90%-180% of the IC₅₀ of D2E7, or an IC₅₀ of 80%-175%of the IC₅₀ of D2E7. In other embodiments, a single CDR substitution canresult in the foregoing differences in IC₅₀ as compared to D2E7, whereasan anti-TNF-α antibody of the disclosure can comprise such substitutionand up to 16 additional substitutions as compared to D2E7.

7.5 Reduced Immunogenicity of Anti-TNF-αAntibodies

In certain aspects, the present disclosure provides anti-TNF-αantibodies having reduced immunogenicity as compared to D2E7. Thepresent disclosure also provides anti-TNF-α antibodies having multipleamino acid substitutions in their CDRs as compared to the CDRs of D2E7,wherein at least one substitution reduces the immunogenicity of theantibody as compared to D2E7. In certain embodiments, the reducedimmunogenicity results from one or more amino acid substitutions thatresult in eliminating or mitigating one or more T cell epitopes.

In certain aspects, the anti-TNF-α antibodies of the disclosure havingreduced immunogenicity have comparable or improved biological activityas compared to D2E7, e.g., affinity towards TNF-α or neutralization ofTNF-α activity. Such properties can be tested, for example, by themethods described in Section 7.3 above.

In certain embodiments, the immunogenicity of an TNF-α antibody of thedisclosure is reduced relative to D2E7 antibody. Such antibodiesgenerally have variant sequences relative to the heavy and/or lightchain variable region in regions corresponding to SEQ ID NO:81 and/orSEQ ID NO:82, and/or SEQ ID NO:83. The antibodies will generally haveone, two or three amino acid substitutions in one, two or all threesequences corresponding to SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83,although up to four or five substitutions one, two or all three regionsare contemplated herein.

Exemplary CDR-L1 substitutions yielding antibodies with lowerimmunogenicity as compared to D2E7 are listed in FIG. 17. Antibodies ofthe disclosure can comprise any of the substitutions or combinations ofsubstitutions listed in FIG. 17, and, optionally, one or more additionalsubstitutions, such as the CDR mutations in any of FIGS. 18-31, singlyor in combination.

As used in the present disclosure, the term “reduced immunogenicity”indicates that the variant sequence as compared to SEQ ID NO:81, SEQ IDNO:82 or SEQ ID NO:83 elicits a reduced proliferative response inperipheral blood mononuclear cells as compared to a peptide of SEQ IDNO:81, SEQ ID NO:82, or SEQ ID NO:83, respectively. An exemplaryproliferation assay that can be used to evaluate the proliferativeresponse is set forth in Section 8 below. The reduced proliferativeresponse can be reflected in terms of the percentage of responders, thestimulation index, or both.

In other embodiments, as compared to a peptide having the sequence ofSEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO;83, the variant sequenceresults in at least 25% fewer responders, in at least 30% fewerresponders, in at least 35% fewer responders, in at least 40% fewerresponders, in at least 45% fewer responders, in at least 50% fewerresponders, in at least 60% fewer responders, in at least 65% fewerresponders, in at least 70% fewer responders, in at least 75% fewerresponders, in at least 80% fewer responders, in at least 85% fewerresponders, in at least 90% fewer responders, in at least 95% fewerresponders, 100% fewer responders, or a reduction in responders in arange between any of the foregoing values, e.g., 25%-75% fewerresponders, 50%-90% fewer responders, 60%-100% fewer responders, 70%-90%fewer responders, or the like.

In other embodiments, the variant sequence results in a stimulationindex that is at least 5% less, at least 10% less, at least 15% less, atleast 20% less, at least 25% less, at least 30% less, at least 35% less,or at least 40% less than the stimulation index elicited by a peptide ofSEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO;83, respectively, or results ina stimulation index reduced by a range between any of the foregoingvalues as compared to a peptide of SEQ ID NO:81, SEQ ID NO:82, or SEQ IDNO;83, e.g., 5%-20% less, 10%-30% less, 25%-35% less, 30%-40% less, orthe like.

Exemplary embodiments of anti-TNF-α antibodies with reducedimmunogenicity as compared to D2E7 comprise one or more of the CDRsubstitutions or combinations of substitutions set forth in FIG. 17.

7.6 Antibody Conjugates

The anti-TNF-α antibodies of the disclosure include antibody conjugatesthat are modified, e.g., by the covalent attachment of any type ofmolecule to the antibody, such that covalent attachment does notinterfere with binding to TNF-α.

In certain aspects, an anti-TNF-α antibody of the disclosure can beconjugated to an effector moiety or a label. The term “effector moiety”as used herein includes, for example, antineoplastic agents, drugs,toxins, biologically active proteins, for example enzymes, otherantibody or antibody fragments, synthetic or naturally occurringpolymers, nucleic acids (e.g., DNA and RNA), radionuclides, particularlyradioiodide, radioisotopes, chelated metals, nanoparticles and reportergroups such as fluorescent compounds or compounds which can be detectedby NMR or ESR spectroscopy.

In one example, anti-TNF-α antibodies can be conjugated to an effectormoiety, such as a cytotoxic agent, a radionuclide or drug moiety tomodify a given biological response. The effector moiety can be a proteinor polypeptide, such as, for example and without limitation, a toxin(such as abrin, ricin A, Pseudomonas exotoxin, or Diphtheria toxin), asignaling molecule (such as α-interferon, β-interferon, nerve growthfactor, platelet derived growth factor or tissue plasminogen activator),a thrombotic agent or an anti-angiogenic agent (e.g., angiostatin orendostatin) or a biological response modifier such as a cytokine orgrowth factor (e.g., interleukin-1 (IL-I), interleukin-2 (IL-2),interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), or nerve growthfactor (NGF)).

In another example the effector moieties can be cytotoxins or cytotoxicagents. Examples of cytotoxins and cytotoxic agents include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorabicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof.

Effector moieties also include, but are not limited to, antimetabolites(e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC5 and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, anthramycin (AMC), calicheamicins orduocarmycins), and anti-mitotic agents (e.g., vincristine andvinblastine).

Other effector moieties can include radionuclides such as, but notlimited to, ¹¹¹In and ⁹⁰Y, Lu¹⁷⁷, Bismuth²¹³, Californium²⁵², Iridium¹⁹²and Tungsten¹⁸⁸/Rhenium¹⁸⁸ and drugs such as, but not limited to,alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Techniques for conjugating such effector moieties to antibodies are wellknown in the art (See, e.g., Hellstrom et al., Controlled Drug Delivery,2nd Ed., at pp. 623-53 (Robinson et al., eds., 1987)); Thorpe et al.,1982, Immunol. Rev. 62:119-58 and Dubowchik et al., 1999, Pharmacologyand Therapeutics 83:67-123).

In one example, the antibody or fragment thereof is fused via a covalentbond (e.g., a peptide bond), through the antibody's N-terminus or theC-terminus or internally, to an amino acid sequence of another protein(or portion thereof; for example, at least a 10, 20 or 50 amino acidportion of the protein). The antibody, or fragment thereof, can linkedto the other protein at the N-terminus of the constant domain of theantibody. Recombinant DNA procedures can be used to create such fusions,for example as described in WO 86/01533 and EP0392745. In anotherexample the effector molecule can increase half-life in vivo, and/orenhance the delivery of an antibody across an epithelial barrier to theimmune system. Examples of suitable effector molecules of this typeinclude polymers, albumin, albumin binding proteins or albumin bindingcompounds such as those described in WO 2005/117984.

In certain aspects, an anti-TNF-α antibody is conjugated to a smallmolecule toxin. In certain exemplary embodiments, an anti-TNF-α antibodyof the disclosure is conjugated to a dolastatin or a dolostatin peptidicanalogs or derivatives, e.g., an auristatin (U.S. Pat. Nos. 5,635,483and 5,780,588). The dolastatin or auristatin drug moiety may be attachedto the antibody through its N (amino) terminus, C (carboxyl) terminus orinternally (WO 02/088172). Exemplary auristatin embodiments include theN-terminus linked monomethylauristatin drug moieties DE and DF, asdisclosed in U.S. Pat. No. 7,498,298, which is hereby incorporated byreference in its entirety (disclosing, e.g., linkers and methods ofpreparing monomethylvaline compounds such as MMAE and MMAF conjugated tolinkers).

In other exemplary embodiments, small molecule toxins include but arenot limited to calicheamicin, maytansine (U.S. Pat. No. 5,208,020),trichothene, and CC1065. In one embodiment of the disclosure, theantibody is conjugated to one or more maytansine molecules (e.g., about1 to about 10 maytansine molecules per antibody molecule). Maytansinemay, for example, be converted to May-SS-Me which may be reduced toMay-SH3 and reacted with an antibody (Chari et al., 1992, CancerResearch 52: 127-131) to generate a maytansinoid-antibody ormaytansinoid-Fc fusion conjugate. Structural analogues of calicheamicinthat can also be used include but are not limited to γ₁ ¹, γ₃ ¹, γ₃ ¹N-acetyl-γ₁ ¹, PSAG, and θ₁ ¹, (Hinman et al., 1993, Cancer Research53:3336-3342; Lode et al., 1998, Cancer Research 58:2925-2928; U.S. Pat.No. 5,714,586; U.S. Pat. No. 5,712,374; U.S. Pat. No. 5,264,586; U.S.Pat. No. 5,773,001).

Antibodies of the disclosure can also be conjugated to liposomes fortargeted delivery (See, e.g., Park et al., 1997, Adv. Pharmacol.40:399-435; Marty & Schwendener, 2004, Methods in Molecular Medicine109:389-401).

In one example antibodies of the present disclosure can be attached topoly(ethyleneglycol) (PEG) moieties. In one particular example theantibody is an antibody fragment and the PEG moieties can be attachedthrough any available amino acid side-chain or terminal amino acidfunctional group located in the antibody fragment, for example any freeamino, imino, thiol, hydroxyl or carboxyl group. Such amino acids canoccur naturally in the antibody fragment or can be engineered into thefragment using recombinant DNA methods. See, for example, U.S. Pat. No.5,219,996. Multiple sites can be used to attach two or more PEGmolecules. PEG moieties can be covalently linked through a thiol groupof at least one cysteine residue located in the antibody fragment. Wherea thiol group is used as the point of attachment, appropriatelyactivated effector moieties (for example, thiol selective derivativessuch as maleimides and cysteine derivatives) can be used.

In a specific example, an anti-TNF-α antibody conjugate is a modifiedFab′ fragment which is PEGylated, i.e., has PEG (poly(ethyleneglycol))covalently attached thereto, e.g., according to the method disclosed inEP0948544. See also Poly(ethyleneglycol) Chemistry, Biotechnical andBiomedical Applications, (J. Milton Harris (ed.), Plenum Press, NewYork, 1992); Poly(ethyleneglycol) Chemistry and Biological Applications,(J. Milton Harris and S. Zalipsky, eds., American Chemical Society,Washington D.C., 1997); and Bioconjugation Protein Coupling Techniquesfor the Biomedical Sciences, (M. Aslam and A. Dent, eds., GrovePublishers, New York, 1998); and Chapman, 2002, Advanced Drug DeliveryReviews 54:531-545. PEG can be attached to a cysteine in the hingeregion. In one example, a PEG-modified Fab′ fragment has a maleimidegroup covalently linked to a single thiol group in a modified hingeregion. A lysine residue can be covalently linked to the maleimide groupand to each of the amine groups on the lysine residue can be attached amethoxypoly(ethyleneglycol) polymer having a molecular weight ofapproximately 20,000 Da. The total molecular weight of the PEG attachedto the Fab′ fragment can therefore be approximately 40,000 Da.

The word “label” when used herein refers to a detectable compound orcomposition which can be conjugated directly or indirectly to ananti-TNF-α antibody of the disclosure. The label can itself bedetectable (e.g., radioisotope labels or fluorescent labels) or, in thecase of an enzymatic label, can catalyze chemical alteration of asubstrate compound or composition which is detectable. Usefulfluorescent moieties include, but are not limited to, fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and thelike. Useful enzymatic labels include, but are not limited to, alkalinephosphatase, horseradish peroxidase, glucose oxidase and the like.

Additional anti-TNF-α antibody conjugates that are useful for, interalia, diagnostic purposes, are described in Section 7.7 below.

7.7 Diagnostic Uses of Anti-TNF-α Antibodies

The anti-TNF-α antibodies of the disclosure, including those antibodiesthat have been modified, e.g., by biotinylation, horseradish peroxidase,or any other detectable moiety (including those described in Section7.6), can be advantageously used for diagnostic purposes.

In particular, the anti-TNF-α antibodies can be used, for example, butnot limited to, to purify or detect TNF-α, including both in vitro andin vivo diagnostic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofTNF-α in biological samples. See, e.g., Harlow et al., Antibodies: ALaboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press,1988), which is incorporated by reference herein in its entirety. In oneembodiment, the anti-TNF-α antibodies of the disclosure can be used fordetecting and quantitating levels of TNF-α in the serum.

The present disclosure further encompasses antibodies or fragmentsthereof conjugated to a diagnostic agent. The antibodies can be useddiagnostically, for example, to detect expression of a target ofinterest in specific cells, tissues, or serum; or to monitor thedevelopment or progression of an immunologic response as part of aclinical testing procedure to, e.g., determine the efficacy of a giventreatment regimen. Detection can be facilitated by coupling the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, radioactive materials, positronemitting metals using various positron emission tomographies, andnonradioactive paramagnetic metal ions. The detectable substance can becoupled or conjugated either directly to the antibody (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. Examples ofenzymatic labels include luciferases (e.g., firefly luciferase andbacterial luciferase; U.S. Pat. No. 4,737,456), luciferin,2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidasesuch as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme,saccharide oxidases (e.g., glucose oxidase, galactose oxidase, andglucose-6-phosphate dehydrogenase), heterocyclic oxidases (such asuricase and xanthine oxidase), lactoperoxidase, microperoxidase, and thelike. Examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

The disclosure provides for the detection of expression of TNF-α,comprising contacting a biological sample (cells, tissue, or body fluidof an individual) using one or more anti-TNF-α antibodies of thedisclosure (optionally conjugated to detectable moiety), and detectingwhether or not the sample is positive for TNF-α expression, or whetherthe sample has altered (e.g., reduced or increased) expression ascompared to a control sample.

Diseases that can be diagnosed using the present methods include, butare not limited to, the diseases described herein. In certainembodiments, the tissue or body fluid is peripheral blood, peripheralblood leukocytes, biopsy tissues such as lung or skin biopsies, andtissue.

7.8 Therapeutic Methods Using Anti-TNF-αAntibodies

7.8.1 Clinical Benefits

The TNF-α antibodies of the present disclosure are useful for treatingdisorders or symptoms of various immune and autoimmune pathologies aswell as inflammatory diseases. TNF-α-related pathologies and diseasesthat can be treated with the anti-TNF-α antibodies of the disclosureinclude, but are not limited to, the following:

-   -   Acute and chronic immune and autoimmune pathologies, such as        systemic lupus erythematosus, rheumatoid arthritis, thyroidosis,        graft versus host disease, scleroderma, diabetes mellitus,        Grave's disease, and the like;    -   Infections, including, but not limited to, sepsis syndrome,        cachexia, circulatory collapse and shock resulting from acute or        chronic bacterial infection, acute and chronic parasitic and/or        bacterial, viral or fungal infectious diseases, such as AIDS        (including sequelae such as cachexia, autoimmune disorders, AIDS        dementia complex and infections);    -   Inflammatory diseases, such as chronic inflammatory pathologies        and vascular inflammatory pathologies, including chronic        inflammatory pathologies such as sarcoidosis, chronic        inflammatory bowel disease, ulcerative colitis, and Crohn's        pathology and vascular inflammatory pathologies, such as, but        not limited to, disseminated intravascular coagulation,        atherosclerosis, and Kawasaki's pathology;    -   Neurodegenerative diseases, including, but not limited to,        demyelinating diseases, such as multiple sclerosis and acute        transverse myelitis; extrapyramidal and cerebellar disorders'        such as lesions of the corticospinal system; disorders of the        basal ganglia or cerebellar disorders; hyperkinetic movement        disorders such as Huntington's Chorea and senile chorea,        drug-induced movement disorders, such as those induced by drugs        which block the CNS, dopamine receptors; hypokinetic movement        disorders, such as Parkinson's disease; Progressive supranucleo        palsy, Cerebellar and Spinocerebellar Disorders, such as        astructural lesions of the cerebellum; spinocerebellar        degenerations (spinal ataxia, Friedreich's ataxia, cerebellar        cortical degenerations, multiple systems degenerations (Mencel,        Dejerine-Thomas, Shi-Drager, and Machado-Joseph); and systemic        disorders (Refsum's disease, abetalipoprotemia, ataxia,        telangiectasia, and mitochondrial multi. system disorder);        demyelinating core disorders, such as multiple sclerosis, acute        transverse myelitis; disorders of the motor unit, such as        neurogenic muscular atrophies (anterior horn cell degeneration,        such as amyotrophic lateral sclerosis, infantile spinal muscular        atrophy and juvenile spinal muscular atrophy); Alzheimer's        disease; Down's Syndrome in middle age; Diffuse Lewy body        disease; Senile Dementia of Lewy body type, Wernicke-Korsakoff        syndrome; chronic alcoholism; Creutzfeldt-Jakob disease;        subacute sclerosing panencephalitis, Hallerrorden-Spatz        disease,- and Dementia pugilistica, or any subset thereof;    -   Malignant pathologies involving TNF-αsecreting tumors or other        malignancies involving TNF-α, such as, but not limited to        leukemias (acute, chronic myelocytic, chronic lymphocytic and/or        myelodyspastic syndrome); lymphomas (Hodgkin's and non-Hodgkin's        lymphomas, such as malignant lymphomas (Burkitt's lymphoma or        Mycosis fungoides), and    -   Alcohol-induced hepatitis.

In certain specific embodiments, the antibodies of the disclosure areused to treat one or more of:

-   -   Moderate to severe rheumatoid arthritis (RA) in adults.    -   Moderate to severe polyarticular juvenile idiopathic arthritis        (JIA) in children 4 years of age and older.    -   Psoriatic arthritis (PsA) in adults.    -   Ankylosing spondylitis (AS) in adults.    -   Moderate to severe Crohn's disease (CD) in adults who have not        responded well to conventional treatments.    -   Moderate to severe chronic plaque psoriasis (Ps) in adults.

Accordingly, the present disclosure provides methods of treating any ofthe foregoing diseases in a patient in need thereof, comprising:administering to the patient an anti-TNF-α antibody of the disclosure.Optionally, said administration is repeated, e.g., after one day, twodays, three days, five days, one week, two weeks, three weeks, onemonth, five weeks, six weeks, seven weeks, eight weeks, two months, orthree months. The repeated administration can be at the same dose or ata different dose. The administration can be repeated once, twice, threetimes, four times, five times, six times, seven times, eight times, ninetimes, ten times, or more. For example, according to certain dosageregimens a patient receives anti-TNF-α therapy for a prolonged period oftime, e.g., 6 months, 1 year or more. The amount of anti-TNF-α antibodyadministered to the patient is in certain embodiments a therapeuticallyeffective amount. As used herein, a “therapeutically effective” amountof TNF-α antibody can be administered as a single dose or over thecourse of a therapeutic regimen, e.g., over the course of a week, twoweeks, three weeks, one month, three months, six months, one year, orlonger. Exemplary therapeutic regimens are described in Section 7.11below.

According to the present disclosure, treatment of a disease encompassesthe treatment of patients already diagnosed as having any form of thedisease at any clinical stage or manifestation; the delay of the onsetor evolution or aggravation or deterioration of the symptoms or signs ofthe disease; and/or preventing and/or reducing the severity of thedisease.

A “subject” or “patient” to whom the anti-TNF-α antibody of thedisclosure is administered is preferably a mammal such as a non-primate(e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkeyor human). In certain embodiments, the subject or patient is a human. Incertain aspects, the human is a pediatric patient. In other aspects, thehuman is an adult patient.

7.9 Pharmaceutical Compositions and Routes of Administration

Compositions comprising an anti-TNF-α antibody of the disclosure and,optionally one or more additional therapeutic agents, such as thecombination therapeutic agents described in Section 7.10 below, areprovided herein. The compositions will usually be supplied as part of asterile, pharmaceutical composition that will normally include apharmaceutically acceptable carrier. This composition can be in anysuitable form (depending upon the desired method of administering it toa patient).

The anti-TNF-α antibodies of the disclosure can be administered to apatient by a variety of routes such as orally, transdermally,subcutaneously, intranasally, intravenously, intramuscularly,intraocularly, topically, intrathecally and intracerebroventricularly.The most suitable route for administration in any given case will dependon the particular antibody, the subject, and the nature and severity ofthe disease and the physical condition of the subject.

For treatment of indications described herein, the effective dose of ananti-TNF-α antibody of the disclosure can range from about 0.001 toabout 75 mg/kg per single (e.g., bolus) administration, multipleadministrations or continuous administration, or to achieve a serumconcentration of 0.01-5000 μg/mL serum concentration per single (e.g.,bolus) administration, multiple administrations or continuousadministration, or any effective range or value therein depending on thecondition being treated, the route of administration and the age, weightand condition of the subject. In a certain embodiment, each dose canrange from about 0.5 μg to about 50 μg per kilogram of body weight, forexample from about 3 μg to about 30 μg per kilogram body weight. Theantibody can be formulated as an aqueous solution and administered bysubcutaneous injection.

Pharmaceutical compositions can be conveniently presented in unit doseforms containing a predetermined amount of an anti-TNF-α antibody of thedisclosure per dose. Such a unit can contain for example but withoutlimitation 5 mg to 5 g, for example 10 mg to 1 g, or 20 to 50 mg.Pharmaceutically acceptable carriers for use in the disclosure can takea wide variety of forms depending, e.g., on the condition to be treatedor route of administration.

Therapeutic formulations of the anti-TNF-α antibodies of the disclosurecan be prepared for storage as lyophilized formulations or aqueoussolutions by mixing the antibody having the desired degree of puritywith optional pharmaceutically-acceptable carriers, excipients orstabilizers typically employed in the art (all of which are referred toherein as “carriers”), i.e., buffering agents, stabilizing agents,preservatives, isotonifiers, non-ionic detergents, antioxidants, andother miscellaneous additives. See, Remington's Pharmaceutical Sciences,16th edition (Osol, ed. 1980). Such additives must be nontoxic to therecipients at the dosages and concentrations employed.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They can be present at concentration rangingfrom about 2 mM to about 50 mM. Suitable buffering agents for use withthe present disclosure include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additionally, phosphate buffers, histidinebuffers and trimethylamine salts such as Tris can be used.

Preservatives can be added to retard microbial growth, and can be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present disclosure include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalconium halides (e.g., chloride, bromide, and iodide),hexamethonium chloride, and alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol.Isotonicifiers sometimes known as “stabilizers” can be added to ensureisotonicity of liquid compositions of the present disclosure and includepolhydric sugar alcohols, for example trihydric or higher sugaralcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol andmannitol. Stabilizers refer to a broad category of excipients which canrange in function from a bulking agent to an additive which solubilizesthe therapeutic agent or helps to prevent denaturation or adherence tothe container wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thio sulfate; low molecular weight polypeptides (e.g.,peptides of 10 residues or fewer); proteins such as human serum albumin,bovine serum albumin, gelatin or immunoglobulins; hydrophylic polymers,such as polyvinylpyrrolidone monosaccharides, such as xylose, mannose,fructose, glucose; disaccharides such as lactose, maltose, sucrose andtrisaccacharides such as raffinose; and polysaccharides such as dextran.Stabilizers can be present in the range from 0.1 to 10,000 weights perpart of weight active protein.

Non-ionic surfactants or detergents (also known as “wetting agents”) canbe added to help solubilize the therapeutic agent as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stressedwithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188etc.), Pluronic polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20,TWEEN®-80, etc.). Nonionic surfactants can be present in a range ofabout 0.05 mg/mL to about 1.0 mg/mL, for example about 0.07 mg/mL toabout 0.2 mg/mL.

Additional miscellaneous excipients include bulking agents (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine, vitamin E), and cosolvents. Further formulationssuitable for the anti-TNF-α antibodies of the disclosure are disclosedin U.S. Pat. App. No. 2004/0033228 A1, the contents of which areincorporated by reference herein in their entirety.

The formulation herein can also contain a combination therapeutic agentin addition to the anti-TNF-α antibody of the disclosure. Examples ofsuitable combination therapeutic agents are provided in Section 7.10below.

The dosing schedule for subcutaneous administration can vary from onceevery six months, five months, four months, three months, two months,once a month to biweekly, weekly, or daily depending on a number ofclinical factors, including the type of disease, severity of disease,and the patient's sensitivity to the anti-TNF-α antibody.

The dosage of an anti-TNF-α antibody of the disclosure to beadministered of will vary according to the particular antibody, the typeof autoimmune or inflammatory disease, the subject, and the nature andseverity of the disease, the physical condition of the subject, thetherapeutic regimen (e.g., whether a combination therapeutic agent isused), and the selected route of administration; the appropriate dosagecan be readily determined by a person skilled in the art.

For the treatment and/or prophylaxis of autoimmune or inflammatorydisease in humans and animals, pharmaceutical compositions comprisinganti-TNF-α antibodies can be administered to patients (e.g., humansubjects) at therapeutically or prophylactically effective dosages(e.g., dosages which result in inhibition of an autoimmune orinflammatory disease and/or relief of autoimmune or inflammatory diseasesymptoms) using any suitable route of administration, such as injectionand other routes of administration known in the art for antibody-basedclinical products.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of an anti-TNF-α antibody ofthe disclosure will be determined by the nature and extent of thecondition being treated, the form, route and site of administration, andthe age and condition of the particular subject being treated, and thata physician will ultimately determine appropriate dosages to be used.This dosage can be repeated as often as appropriate. If side effectsdevelop the amount and/or frequency of the dosage can be altered orreduced, in accordance with normal clinical practice.

7.10 Combination Therapy

Described below are combinatorial methods in which the anti-TNF-αantibodies of the disclosure can be utilized. The combinatorial methodsof the disclosure involve the administration of at least two agents to apatient, the first of which is an anti-TNF-α antibody of the disclosure,and the additional agent(s) is a combination therapeutic agent. Theanti-TNF-α antibody and the combination therapeutic agent(s) can beadministered simultaneously, sequentially or separately.

The combinatorial therapy methods of the present disclosure can resultin a greater than additive effect, providing therapeutic benefits whereneither the anti-TNF-α antibody or combination therapeutic agentadministered in an amount that is alone therapeutically effective.

In the present methods, the anti-TNF-α antibody of the disclosure andthe combination therapeutic agent can be administered concurrently,either simultaneously or successively. As used herein, the anti-TNF-αantibody of the disclosure and the combination therapeutic agent aresaid to be administered successively if they are administered to thepatient on the same day, for example during the same patient visit.Successive administration can occur 1, 2, 3, 4, 5, 6, 7 or 8 hoursapart. In contrast, the anti-TNF-α antibody of the disclosure and thecombination therapeutic agent are said to be administered separately ifthey are administered to the patient on the different days, for example,the anti-TNF-αantibody of the disclosure and the combination therapeuticagent can be administered at a 1-day, 2-day or 3-day, one-week, 2-weekor monthly intervals. In the methods of the present disclosure,administration of the anti-TNF-α antibody of the disclosure can precedeor follow administration of the combination therapeutic agent.

As a non-limiting example, the anti-TNF-α antibody of the disclosure andcombination therapeutic agent can be administered concurrently for aperiod of time, followed by a second period of time in which theadministration of the anti-TNF-α antibody of the disclosure and thecombination therapeutic agent is alternated.

Because of the potentially synergistic effects of administering ananti-TNF-αantibody of the disclosure and a combination therapeuticagent, such agents can be administered in amounts that, if one or bothof the agents is administered alone, is/are not therapeuticallyeffective.

In certain aspects, the combination therapeutic agent is ananti-rheumatic drug, an anti-inflammatory agent, a chemotherapeuticagent, a radiotherapeutic, an immunosuppressive agent, or a cytotoxicdrug.

Anti-rheumatic drugs include, but are not limited to, auranofin,azathioprine, chloroquine, D-penicillamine, gold sodium thiomalatehydroxychloroquine, Myocrisin and sulfasalzine methotrexate.

Anti-inflammatory agents include, but are not limited to, dexamethasone,pentasa, mesalazine, asacol, codeine phosphate, benorylate, fenbufen,naprosyn, diclofenac, etodolac and indomethacin, aspirin and ibuprofen.

Chemotherapeutic agents include, but are not limited to, radioactivemolecules, toxins, also referred to as cytotoxins or cytotoxic agents,which includes any agent that is detrimental to the viability of cells,agents, and liposomes or other vesicles containing chemotherapeuticcompounds. Examples of suitable chemotherapeutic agents include but arenot limited to 1-dehydrotestosterone, 5-fluorouracil decarbazine,6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin,alkylating agents, allopurinol sodium, altretamine, amifostine,anastrozole, anthramycin (AMC)), anti-mitotic agents, cisdichlorodiamineplatinum (II) (DDP) cisplatin), diamino dichloro platinum,anthracyclines, antibiotics, antimetabolites, asparaginase, BCG live(intravesical), betamethasone sodium phosphate and betamethasoneacetate, bicalutamide, bleomycin sulfate, busulfan, calcium leucouorin,calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine(BSNU), Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugatedestrogens, Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine,cytochalasin B, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin(formerly actinomycin), daunirubicin HCL, daunorucbicin citrate,denileukin diftitox, Dexrazoxane, Dibromomannitol, dihydroxy anthracindione, Docetaxel, dolasetron mesylate, doxorubicin HCL, dronabinol, E.coli L-asparaginase, eolociximab, emetine, epoetin-α, ErwiniaL-asparaginase, esterified estrogens, estradiol, estramustine phosphatesodium, ethidium bromide, ethinyl estradiol, etidronate, etoposidecitrororum factor, etoposide phosphate, filgrastim, floxuridine,fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinicacid, gemcitabine HCL, glucocorticoids, goserelin acetate, gramicidin D,granisetron HCL, hydroxyurea, idarubicin HCL, ifosfamide, interferonα-2b, irinotecan HCL, letrozole, leucovorin calcium, leuprolide acetate,levamisole HCL, lidocaine, lomustine, maytansinoid, mechlorethamine HCL,medroxyprogesterone acetate, megestrol acetate, melphalan HCL,mercaptipurine, mesna, methotrexate, methyltestosterone, mithramycin,mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate,ondansetron HCL, paclitaxel, pamidronate disodium, pentostatin,pilocarpine HCL, plimycin, polifeprosan 20 with carmustine implant,porfimer sodium, procaine, procarbazine HCL, propranolol, rituximab,sargramostim, streptozotocin, tamoxifen, taxol, teniposide, tenoposide,testolactone, tetracaine, thioepa chlorambucil, thioguanine, thiotepa,topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin,vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

In yet other aspects of the disclosure, the combination therapeuticagent is a TNF-α antagonist other than the anti-TNF-α antibody of thedisclosure. Examples of such TNF-αantagonists include, but are notlimited to, soluble TNF-α receptors; etanercept (ENBREL™; Immunex) or afragment, derivative or analog thereof; infliximab (REMICADE®; Centacor)or a derivative, analog or antigen-binding fragment thereof; IL-10,which is known to block TNF-α production via interferon-γ-activatedmacrophages (Oswald et al., 1992, Proc. Natl. Acad. Sci. USA89:8676-8680), TNFR-IgG (Ashkenazi et al., 1991, Proc. Natl. Acad. Sci.USA 88:10535-10539); the murine product TBP-1 (Serono/Yeda); the vaccineCytoTAb (Protherics); antisense molecule 104838 (ISIS); the peptideRDP-58 (SangStat); thalidomide (Celgene); CDC-801 (Celgene); DPC-333(Dupont); VX-745 (Vertex); AGIX-4207 (AtheroGenics); ITF-2357(Italfarmaco); NPI-13021-31 (Nereus); SCIO-469 (Scios); TACE targeter(Immunix/AHP); CLX-120500 (Calyx); Thiazolopyrim (Dynavax); auranofin(Ridaura) (SmithKline Beecham Pharmaceuticals); quinacrine (mepacrinedichlorohydrate); tenidap (Enablex); Melanin (Large Scale Biological);and anti-p38 MAPK agents by Uriach.

Additional second therapeutic agents useful in combination with ananti-TNF-α antibody and particular indications for which combinationtherapy with such second therapeutic agents are useful are disclosed inWO 2004/004633, which is incorporated by reference herein in itsentirety.

7.11 Therapeutic Regimens

The present disclosure provides therapeutic regimens involving theadministration of the anti-TNF-α antibodies of the disclosure. Thetherapeutic regimen will vary depending on the patient's age, weight,and disease condition. The therapeutic regimen can continue for 2 weeksto indefinitely. In specific embodiments, the therapeutic regimen iscontinued for 2 weeks to 6 months, from 3 months to 5 years, from 6months to 1 or 2 years, from 8 months to 18 months, or the like. Thetherapeutic regimen can be a non-variable dose regimen or amultiple-variable dose regimen, for example as described in WO2005/110452, which is incorporated by reference in its entirety.

For the dosage exemplary regimens described below, the anti-TNF-αantibody can be administered as a sterile, preservative-free solutionfor subcutaneous administration.

In certain embodiments, the drug product is supplied as either asingle-use, prefilled pen within which is enclosed a 1 mL prefilledglass syringe, or as a single-dose, 1 mL prefilled glass syringe. Foradult patients, in certain embodiments the syringe delivers 0.8 mL of apharmaceutically acceptable solution comprising the anti-TNF-α antibodyof the disclosure. In a specific embodiment, in addition to the antibodythe solution contains 4.93 mg sodium chloride, 0.69 mg monobasic sodiumphosphate dihydrate, 1.22 mg dibasic sodium phosphate dihydrate, 0.24 mgsodium citrate, 1.04 mg citric acid monohydrate, 9.6 mg mannitol, 0.8 mgpolysorbate 80, and water for injection (USP) with sodium hydroxideadded as necessary to adjust pH. For pediatric patients, in certainembodiments the syringe delivers 0.4 mL of a pharmaceutically acceptablesolution comprising the anti-TNF-αantibody of the disclosure. In aspecific embodiment, in addition to the antibody the solution contains2.47 mg sodium chloride, 0.34 mg monobasic sodium phosphate dihydrate,0.61 mg dibasic sodium phosphate dihydrate, 0.12 mg sodium citrate, 0.52mg citric acid monohydrate, 4.8 mg mannitol, 0.4 mg polysorbate 80, andwater for injection (USP) with sodium hydroxide added as necessary toadjust pH.

For treatment rheumatoid arthritis, psoriatic arthritis, and ankylosingspondylitis, an anti-TNF-α antibody of the disclosure can beadministered at a dose of 10 to 50 mg (e.g., 10 mg, 15 mg, 20 mg, 25 mg,30 mg, 35 mg, 40 mg, 45 mg or 50 mg) every other week. Methotrexate,glucocorticoids, salicylates, nonsteroidal anti-inflammatory drugs(NSAIDs), analgesics or other disease-modifying antirheumatics drug(DMARDs) can be continued during treatment with the anti-TNF-α antibodyof the disclosure. In rheumatoid arthritis, some patients not takingconcomitant methotrexate can derive additional benefit from increasingthe dosing frequency from biweekly to weekly.

For treatment of juvenile idiopathic arthritis, an anti-TNF-α antibodyof the disclosure is administered at a dose that depends on thepatient's weight. In certain non-limiting embodiments, the dose forpediatric patients weighing 15 kg (33 lbs) to under 30 kg (66 lbs)ranges from 5 to 25 mg (e.g., 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 20mg, or 25 mg) every other week. In certain non-limiting embodiments, thedose for pediatric patients weighing greater than 30 kg (66 lbs) rangesfrom 10 to 50 mg (e.g., 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg,45 mg or 50 mg) every other week. Methotrexate, glucocorticoids,salicylates, NSAIDs or analgesics can be continued during treatment withthe anti-TNF-α antibody.

For treatment of Crohn's Disease, an anti-TNF-α antibody of thedisclosure can be administered in certain non-limiting embodiments at adose of 40-280 mg (e.g., 40 mg, 80 mg, 100 mg, 120 mg, 140 mg, 160 mg,180 mg, 200 mg, 240 mg, or 280 mg) given initially (on Day 1 or dividedbetween Day 1 and Day 2), followed by a dose of approximately 40% to 60%(e.g., 50%) of the initial dose two weeks later (Day 15). Two weekslater (Day 29), a maintenance dose of 20% to 30% (e.g., 25%) of theinitial dose is administered every other week. Aminosalicylates,corticosteroids, and/or immunomodulatory agents (e.g., 6-mercaptopurineand azathioprine) can be continued during treatment with the anti-TNF-αantibody.

For treatment of plaque psoriasis, an anti-TNF-α antibody of thedisclosure can be administered in certain non-limiting embodiments at adose of 40-160 mg (e.g., 40 mg, 80 mg, 100 mg, 120 mg, 140 mg, or 160 mggiven initially followed by half the initial dose given every other weekstarting one week after the initial dose.

7.12 Diagnostic and Pharmaceutical Kits

Encompassed by the present disclosure are pharmaceutical kits containingthe anti-TNF-α antibodies (including antibody conjugates) of thedisclosure. The pharmaceutical kit is a package comprising theanti-TNF-α antibody of the disclosure (e.g., either in lyophilized formor as an aqueous solution) and one or more of the following:

-   -   A combination therapeutic agent, for example as described in        Section 7.10 above;    -   A device for administering the anti-TNF-α antibody, for example        a pen, needle and/or syringe; and    -   Pharmaceutical grade water or buffer to resuspend the antibody        if the antibody is in lyophilized form.

In certain aspects, each unit dose of the anti-TNF-α antibody ispackaged separately, and a kit can contain one or more unit doses (e.g.,two unit doses, three unit doses, four unit doses, five unit doses,eight unit doses, ten unit doses, or more). In a specific embodiment,the one or more unit doses are each housed in a syringe or pen.

Diagnostic kits containing the anti-TNF-α antibodies (including antibodyconjugates) of the disclosure are also encompassed herein. Thediagnostic kit is a package comprising the anti-TNF-α antibody of thedisclosure (e.g., either in lyophilized form or as an aqueous solution)and one or more reagents useful for performing a diagnostic assay. Wherethe anti-TNF-α antibody is labeled with an enzyme, the kit can includesubstrates and cofactors required by the enzyme (e.g., a substrateprecursor which provides the detectable chromophore or fluorophore). Inaddition, other additives can be included, such as stabilizers, buffers(e.g., a block buffer or lysis buffer), and the like. In certainembodiments, the anti-TNF-α antibody included in a diagnostic kit isimmobilized on a solid surface, or a solid surface (e.g., a slide) onwhich the antibody can be immobilized is included in the kit. Therelative amounts of the various reagents can be varied widely to providefor concentrations in solution of the reagents which substantiallyoptimize the sensitivity of the assay. In a specific embodiment, theantibody and one or more reagents can be provided (individually orcombined) as dry powders, usually lyophilized, including excipientswhich on dissolution will provide a reagent solution having theappropriate concentration.

8. EXAMPLE 1 Identification of Deimmunized Variants of D2E7 8.1Materials & Methods

8.1.1 Peptides

Peptides were synthesized using a multi-pin format by Mimotopes(Adelaide,

-   Australia). The sequences of the D2E7 light and heavy chain V    regions were synthesized as 15-mer peptides overlapping by 12 amino    acids (FIG. 1 and FIG. 7) for a total of 69 peptides. Peptides    arrived lyophilized and were re-suspended in DMSO (Sigma-Aldrich) at    approximately 1-2 mg/mL. Stock peptides were kept frozen at −20° C.

8.1.2 Human Peripheral Blood Mononuclear Cells

Community donor buffy coat products were purchased from the StanfordBlood Center, Palo Alto, Calif. Buffy coat material was diluted 1:1(v:v) with DPBS containing no calcium or magnesium. Diluted buffy coatmaterial (25-35 mL) was underlayed in 50 mL conical centrifuge tubes(Sarsted or Costar) with 12.5 mL mL of FicollPaque-PLUS (GE Healthcare).The samples were centrifuged at 900 g for 30 minutes at roomtemperature. Peripheral blood mononuclear cells (PBMC) were collectedfrom the interface. DPBS was added to bring the final volume to 50 mLmLand the cells were centrifuged at 350 g for 5 minutes. Pelleted cellswere resuspended in DPBS and counted.

8.1.3 Dendritic Cells

For isolation of dendritic cells, T75 culture flasks (Costar) wereseeded with 10⁸ freshly isolated PBMC in a total volume of 30 mL AIM Vmedia (Invitrogen). Excess PBMC were frozen at −80° C. in 90% fetal calfserum (FCS), 10% DMSO at 5×10⁷ cells/ml. T75 flasks were incubated at37° C. in 5% CO₂ for 2 hours. Nonadherent cells were removed, and theadherent monolayer was washed with DPBS. To differentiate dendriticcells from monocytes, 30 mL of AIM V media containing 800 units/mL ofGM-CSF (R and D Systems) and 500 units/mL IL-4 (R and D Systems) wereadded. Flasks were incubated for 5 days. On day 5 IL-1α (Endogen) andTNF-α (Endogen) were added to 50 pg/mL and 0.2 ng/ml. Flasks wereincubated for two more days. On day 7, dendritic cells were collected bythe addition of 3 mL of 100 mM EDTA containing 0.5 to 1.0 mg Mitomycin C(Sigma-Aldrich) for a final concentration of 10 mM EDTA and 16.5 to 33μg/mL Mitomycin C. Alternatively, dendritic cells can be irradiated with4,000 rads for fixation. Flasks were incubated an additional hour at 37°C. and 5% CO₂. Dendritic cells were collected, and washed in AIM V media2-3 times.

8.1.4 Cell Culture

On day 7, previously frozen autologous PBMC were thawed quickly in a 37°C. water bath. Cells were immediately diluted into DPBS or AIM V mediaand centrifuged at 350 g for 5 minutes. CD4+ cells were enriched bynegative selection using magnetic beads (Easy-Sep CD4+ kit, Stem CellTechnologies). Autologous CD4+ T cells and dendritic cells werecocultured at 2×10⁵ CD4+ T cells per 2×10⁴ dendritic cells per well in96 well round bottomed plates (Costar 9077). Peptides were added atapproximately 5 μg/mL. Control wells contained the DMSO (Sigma) vehiclealone at 0.25% v:v. Positive control wells contained DMSO at 0.25% andtetanus toxoid (List Biologicals or CalBioChem) at 1 μg/mL. Cultureswere incubated for 5 days. On day 5, 0.25 μCi per well of tritiatedthymidine (Amersham or GE Healthcare) was added. Cultures were harvestedon day 6 to filtermats using a Packard Filtermate Cell harvester.Scintillation counting was performed using a Wallac MicroBeta 1450scintillation counter (Perkin Elmer).

8.1.5 Data Analyses

Average background CPM values were calculated by averaging individualresults from 6 to 12 replicates. The CPM values of the four positivecontrol wells were averaged. Replicate or triplicate wells for eachpeptide were averaged. Stimulation index values for the positive controland the peptide wells were calculated by dividing the averageexperimental CPM values by the average control values. In order to beincluded in the dataset, a stimulation index of greater than 3.0 in thetetanus toxoid positive control wells was required. A response was notedfor any peptide resulting in a stimulation index of 2.95 or greater.Peptides were tested using peripheral blood samples from a group of 81donors. Responses to all peptides were compiled. For each peptidetested, the percentage of the donor set that responded with astimulation index of 2.95 or greater was calculated. In addition, theaverage stimulation index for all donors was calculated.

8.1.6 HLA Genotype Analysis

HLA DRB1 and HLA DQB1 alleles were determined for each donor using thecommercially available Dynal RELI typing kits (Invitrogen, UK). Lowstringency SSO results are reported. HLA associations were determinedfor responsiveness to any given peptide using a Chi-squared analysis(one degree of freedom). Where an allele was present in both of theresponder and non-responder populations, a relative risk value wasreported.

8.1.7 Competition ELISA of D2E7 Variant Antibodies

TNF-α was adhered onto a microwell plate, by contacting the plate with asolution of TNF-α at a concentration of 1 μg/mL in PBS over night at 4°C. The plate was washed in 0.1% Tween 20 in PBS and blocked inSuperblock (Thermo Scientific, Rockford, Ill.). A mixture ofsub-saturating amount of biotinylated D2E7 (80 ng/mL) and unlabeled D2E7(the “reference” antibody) or competing anti-TNF-α antibody (the “test”antibody) antibody in serial dilution (at a concentration of 2.8 μg/mL,8.3 μg/mL, or 25 μg/mL) in ELISA buffer (e.g., 1% BSA and 0.1% Tween 20in PBS) was added to wells and plates were incubated for 1 hour withgentle shaking. The plate was washed, 1 μg/mL HRP-conjugatedStreptavidin diluted in ELISA buffer was added to each well and theplates incubated for 1 hour. Plates were washed and bound antibodieswere detected by addition of TMB (Biofx Laboratories Inc., Owings Mills,Md.). The reaction was terminated by addition of stop buffer (e.g., BioFX Stop Reagents, Biofx Laboratories Inc., Owings Mills, Md.) and theabsorbance was measured at 650 nm using microplate reader (e.g.,VERSAmax, Molecular Devices, Sunnyvale, Calif.). The IC₅₀ values werecalculated for each antibody. The experiment was performed three times,and average results are shown as a percent of the parent antibodybinding result.

8.1.8 Bioassay

3×10⁴ murine L929 cells were plated into individual wells of a flatbottomed 96-well microtiter plate. The cells were incubated overnight at37° C. in a humidified 5% CO₂ incubator. The next day, serial dilutionsof the anti-TNF-α antibody (e.g., 0.712 μg/mL, 0.949 μg/mL, 1.27 μg/mL,1.69 μg/mL, 2.25 μg/mL or 3 μg/mL) were prepared in 25 μL of serum-freemedium and added to cells (such that the final concentration in 150 μLculture was 119 ng/mL, 158 ng/mL, 211 ng/mL, 282 ng/mL, 375 ng/mL or 500ng/mL). After a 2-hour incubation at 37° C. in 5% CO₂, 254, of a 240ng/mL solution of TNF-α were added, for a final concentration of 40ng/mL, and the cells were further incubated for 48 hours at 37° C. in 5%CO₂. The wells were scored for cytotoxicity as compared to controlplates, which treated with TNF-α but incubated with an isotype controlantibody or with the parent antibody, D2E7) using a CellTiter-Blueviability assay (Promega, Madison, Wis.). IC₅₀ values were determinedand expressed as percent of the parental D2E7 result.

8.1.9 Kinetic Analysis of D2E7 Variants by BIAcore

Binding affinities of anti-TNF-α antibodies were measured by using aBIAcore 2000 and 3000 surface plasmon resonance system (BIAcore, GEHealthcare, Piscataway, N.J.). Polyclonal goat anti-human Fc antibody(Jackson Immunoresearch) was first immobilized to the biosensor surfaceusing standard BIAcore amine coupling reagents(N-ethyl-N′-dimethylamino-propylcarbodiimide, EDC; N-hydroxysuccinimide,NHS; and ethanolamine HCl, pH 8.5), followed by the capture ofanti-TNF-α antibodies (D2E7 and D2E7 variants) on parallel surfaces at alow flow rate of 5 μL/min. RL was kept low to achieve a low Rmax of25-60 RU. No capture of the antibody was made on the reference surfaceto serve as a negative control. Subsequently, TNF-α was injected to allflow cells at a flow rate of 80 μL/min for three minutes to monitorassociation followed by a 30-minute flow of HBS-P running buffer (10 mMHEPES, 150 mM sodium chloride, 0.005% P-20, pH 7.4) to monitor thedissociation phase. At each cycle, TNF-α (R&D systems, Minneapolis,Minn.), in 6 different concentrations of TNF ranging between 0 nM and128 and at four-fold increments, was injected over the surface. Thesurface was regenerated with 1.5% H₃PO₄ at a flow rate of 100 μL/min intwo brief pulses at the end of each cycle.

The binding kinetics of each TNF-α and antibody pair were calculatedfrom a global analysis of sensorgram data collected from the differentconcentrations of TNF-α using the BIAevaluate program. Doublereferencing was applied in each analysis to eliminate backgroundresponses from the reference surface and buffer only control (0 nM ofTNF-α). The dissociation constants (K_(D)), the association rateconstants (k_(on)) and the dissociation rate constants (k_(off)) of eachbinding pair was obtained by simultaneously fitting the association anddissociation phases of the sensorgram using the 1:1 Langmuir bindingwith mass transfer model. Each set of experiments was performed 3separate times.

8.2 Results

8.2.1 Identification of CD4+ T Cell Epitopes in the D2E7 VH And VLRegions

CD4+ T cell epitope peptides were identified by an analysis of thepercent responses to the peptides within the set of 81 donors. Theaverage percent response and standard deviation were calculated for allpeptides tested describing the D2E7 heavy chain and light chain. Aresponse rate greater than or equal to the average background responseplus three standard deviations was considered a potential CD4+ T cellepitope. For the D2E7 light chain V region, 32 peptides were tested(FIG. 2) which resulted in an average background percent response of5.09+3.53%. Three standard deviations above background was determined tobe 15.68%. One peptide at position 8 displayed this level of response inthe D2E7 light chain peptide dataset, with a response rate of 17.28%(FIG. 2). In addition, the peptide at position 11 displayed a very highresponse rate of 12.35%. For the D2E7 heavy chain V region, 37 peptideswere tested (FIG. 3). The average background percent response was2.64+2.04%. Three standard deviations above background was 8.78%. Onepeptide within the D2E7 heavy chain dataset, #20, achieved a percentresponse of 8.64% (FIG. 3).

The average stimulation index was calculated for all peptides in thedataset. Light chain peptide #8 had a high average stimulation index of1.97+0.08 s.e.m. The peptide at position #11 returned an averagestimulation index of 1.63+0.32 s.e.m. Peptide #27 in the light chaindataset had an average SI of 1.83. This is due to a single donor with anunusually high stimulation index of 29 to this peptide. Heavy chainpeptide #20 had an average stimulation index value of 1.34+0.05 s.e.m.All of these values are significantly higher than the averagestimulation index for all peptides in the two datasets (1.02+0.02 forall 68 heavy chain and light chain peptides).

These data indicate that there are two major CD4+ T cell epitope regionsin D2E7 (FIG. 8). In the VH region, an epitope is found at peptideposition 20 that encompasses the junction of framework 2 and CDR2. InFIG. 8, the CDR-derived amino acids are underlined. In the light chain,a large region that can contain more than one CD4+ T cell epitopeincludes peptides #8 and #11. These peptides span a section of framework1, CDR1 and framework 2 of the light chain.

8.2.2 HLA Associations with Responses to the VL Epitope Peptides

The HLA class II genotypes of all 81 donors in the peptide dataset weredetermined using a low-stringency SSO PCR-based method. Associationsbetween the presence of a particular HLA allele and responses to the twoVL peptides were determined by chi squared analysis. Fischer P valuesand relative risks were determined for all HLA types and both peptides(FIG. 9). There were no significant correlations between any HLA DR orDQ type and a response to VL peptide #8 (T22-Y36). This result suggeststhat the peptide is capable of binding to HLA class II molecules in abroadly promiscuous manner. CD4+ T cell proliferative responses to theVL peptide #11 (N31-K45) were tightly associated with the presence ofHLA-DQ2 (p=0.003; relative risk=7.7). As HLA-DR3 is in linkagedisequilibrium with HLA-DQ2, the association between a response to thispeptide and HLA-DR3 was present but did not reach statisticalsignificance (p=0.10; relative risk 3.3). In addition to HLA-DQ2, asassociation was found between HLA-DR12 and a response to N31-K45(p=0.03; relative risk 5.2). The HLA responses to the VH peptide #20were not tested as there were too few total responders. Since theresponders to the two VL peptides were discrete it can be concluded thatthey represent two separate peptide epitopes. Therefore, the D2E7 VH andVL region contains three prominent peptide epitope regions.

8.2.3 Identification of Reduced Immunogenicity Variants

Alanine Scan Modifications:

A twenty-one amino acid sequence of the D2E7 light chain encompasses theepitopes at T22-Y36 and N31-K45. The twenty one amino acid sequenceselected was C23-K45. Alanine modifications were incorporated at eachamino acid (FIG. 10). A set of 99 donors was tested with the variantpeptides (FIG. 4). The parent 21-mer was created 4 times within thepeptide set. These four replicates serve as a control for thereproducibility of the assay. The average parent peptide response was8.3%, with a CV % of 30%. Therefore, variant peptides with an averagepercent of less than 5.8% could be considered to have a reduced rate ofresponse. The most reduced variants were C23A (2.02%) and P40A (3.03%,see FIG. 4). The cysteine at position 23 is invariant, and is thereforenot a good candidate for modification in the whole protein. Due to theunique nature of proline residues a modification of this residue is alsonot likely to yield a functional variant antibody. The third candidatewould be Y32A (4.04%). Additionally, there are a number of variants thatresulted in an average response rate of 5.05%. These changes could alsobe effective but would need to be tested as whole protein molecules forboth reduced immunogenicity and functional activity.

A set of alanine-modified peptides based on the sequence of the D2E7 VHepitope peptide were also tested (data not shown). The response rate ofthe parent unmodified peptide in the replicate test was very low.Therefore this peptide was no longer studied.

Antigen Binding Study:

The CDR-L1 region of the D2E7 antibody was subjected to comprehensivemutational analysis. Based on antigen-binding studies performed inconjunction with the mutational analyses, a set of candidate amino acidsubstitutions within the CDR-L1 region was identified that did notsignificantly reduce the affinity of the antibody to TNF-α (FIG. 11).Several variant antibodies containing the candidate CDR-L1 substitutionswere analyzed using BIAcore and ELISA (FIG. 12). Peptides were generatedcontaining amino acid modifications within the CDR-L1 region that hadthe property of altering the amino acid sequence while retaining theaffinity of the overall antibody molecule (FIG. 13). The modifiedepitope peptides were tested as single amino-acid modifications, or asdouble modifications. The double modifications contained a glutamine ora glycine at position 32, or a serine or glycine at position 34. A totalof 79 peptides were tested including two syntheses of the parent 21-merpeptide. A total of 102 donors were tested with the variant peptides andthe results are shown in FIG. 5. The average percent response of theparent peptides was 10.3+2.1%. For a percent response rate to be lessthan 3 standard deviations from the parent the response rate would beless than 4%. The average stimulation index for the parent peptides was1.49+0.15. For a stimulation index to reach three standard deviationsbelow the parent response it would be 1.03 or lower.

Subsequent affinity measurements of the Y32G and Y32Q mutations showedthat this amino acid modification had a negative impact on antigenbinding. Therefore, all peptides carrying this modification were removedfrom the analysis. A total of 10 peptides were selected for furtherstudy. All selected peptides had a response rate less than 4%. However,none of the peptides demonstrated stimulation indexes 3 standarddeviations below the average parent response (FIG. 14).

8.2.4 Affinity and Bioactivity Testing of Modified D2E7 VariantAntibodies

Ten variant D2E7 VL region constructs were cloned along with theunmodified VH region into a human IgG₁-containing plasmid, expressed in293T/17 cell lines by transient transfection, and antibodies purified byProtein A or Protein G affinity. The purified antibodies were tested forTNF-α binding in a competition ELISA assay. All ten variants competedfor TNF-α binding with the unmodified D2E7 antibody. However, there wasa range of affinities displayed, from approximately equivalent affinityof the Q27R+A34S variant, to a 10× reduction in affinity of theN31S+A34S variant (FIG. 6).

A TNF-α toxicity bioassay was performed. L292 cells were seeded into 96well plates, and a constant concentration of TNF-α was added to theculture medium. The variant antibodies were titrated into the medium. AnEC₅₀ value was determined for each variant (FIG. 15). Similarly, thevariant Q27R+A34S displayed an EC₅₀ value approximately equivalent tothe parent D2E7 antibody.

Finally, affinity of the antibodies for TNF-α was determined by BIAcoreanalysis (FIG. 16). Of the ten variants tested, the Q27H+A34S, Q27R+A34Sand G28S+A34S variants all displayed association and dissociation ratessimilar to D2E7. The final affinity values for the variants were in the130 pM range as compared to D2E7 with a measured affinity in theseexperiments of 114 pM.

9. EXAMPLE 2 Identification of Variants of D2E7 with Increased Affinityto TNF-α

The D2E7 antibody was subjected to comprehensive mutational analysis toidentify mutants that had increased affinity to TNF-α as compared toD2E7. The increased affinity of candidate mutants to TNF-α was analyzedby ELISA and BIAcore to confirm their characteristics as compared toD2E7.

9.1 Materials & Methods

9.1.1 Competition ELISA

Competition ELISA assays were done as described in Section 8.1.7. ELISAwas repeated twice and average fold improvement in IC₅₀ is shown asWT/x.

9.1.2 BIAcore

BIAcore assays were done as described in Section 8.1.9.

9.2 Results

CDR variants of D2E7 that had improved K_(D) (as measured by BIAcore),improved ability to compete in ELISA, or both relative to D2E7 are shownin FIGS. 18 and 31.

10. SPECIFIC EMBODIMENTS, CITATION OF REFERENCES

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A monoclonal antibody or a binding fragmentthereof which: (a) specifically binds to human TNF-α; (b) competes forbinding to human TNF-α with an antibody comprising a V_(H) sequence ofSEQ ID NO:2 and a V_(L) sequence of SEQ ID NO:4; and (c) comprises sixCDRs that have one or more amino acid substitutions or combinations ofamino acid substitutions as compared to the CDRs of SEQ ID NO:5(CDR-H1), SEQ ID NO:6 (CDR-H2), SEQ ID NO:7 (CDR-H3), SEQ ID NO:8(CDR-L1), SEQ ID NO:9 (CDR-L2) and SEQ ID NO:10 (CDR-L3), wherein saidone or more amino acid substitutions or combinations of substitutionsare selected from: (1) the CDR-L1 substitutions R7Q; A11S; R7Q+A11S;N8T; N8T+A11S; I6T; A11G; I6T+A11G; Q4G; Q4G+A11S; Q4G+A11G; Q4H;Q4H+A11S; Q4R; Q4R+A11S; G5S+A11S; N8S+A11S; I6T+A11S; and N8T+A11G; (2)the CDR-L2 substitutions S3K or S3R; T4H, T4Q, T4F, T4W or T4Y; L5R orL5K; and Q6R; (3) the CDR-H1 substitutions: Y2H and A3G; and (4) theCDR-H2 substitution: T3N.
 2. The monoclonal antibody or binding fragmentof claim 1, which comprises at least one amino acid substitutionselected from S3K or S3R in CDR-L2; T4H, T4Q, T4F, T4W or T4Y in CDR-L2;L5R or L5K in CDR-L2; Q6R in CDR-L2; Y2H in CDR-H1; A3G in CDR-H1 andT3N in CDR-H2.
 3. The monoclonal antibody or binding fragment of claim2, which comprises at least one amino acid substitution selected fromT4F, T4W or T4Y in CDR-L2; L5R or L5K in CDR-L2; Q6R in CDR-L2; Y2H inCDR-H1; A3G in CDR-H1 and T3N in CDR-H2.
 4. The monoclonal antibody orbinding fragment of claim 1, which comprises at least one amino acidsubstitution selected from R7Q in CDR-L1; A11S in CDR-L1; R7Q+A11S inCDR-L1; N8T in CDR-L1; N8T+A11S in CDR-L1; I6T in CDR-L1; A11G inCDR-L1; I6T+A11G in CDR-L1; Q4G in CDR-L1; Q4G+A11S in CDR-L1; Q4G+A11Gin CDR-L1; Q4H in CDR-L1; Q4H+A11S in CDR-L1; Q4R in CDR-L1; Q4R+A11S inCDR-L1; G5S+A11S in CDR-L1; N8S+A11S in CDR-L1; I6T+A11S in CDR-L1; andN8T+A11G in CDR-L1.
 5. The monoclonal antibody or binding fragment ofclaim 1 which is a human or humanized antibody, or binding fragment of ahuman or humanized antibody, respectively.
 6. The monoclonal antibody orbinding fragment of claim 1 which is an IgG.
 7. The monoclonal antibodyor binding fragment of claim 6 which is an IgG₁.
 8. The monoclonalantibody or binding fragment of claim 1 which has the heavy chainframework sequences of the V_(H) sequence of SEQ ID NO:2 and the lightchain framework sequences of the V_(L) sequence of SEQ ID NO:4.
 9. Themonoclonal antibody or binding fragment of claim 1 which is a bispecificantibody or a TNF-α binding fragment of a bispecific antibody.
 10. Themonoclonal antibody or binding fragment of claim 9, wherein saidbispecific antibody is specific to TNF-α and another pro-inflammatorycytokine.
 11. The monoclonal antibody or binding fragment of claim 10,wherein said pro-inflammatory cytokine is lymphotoxin, interferon-γ, orinterleukin-1.
 12. The monoclonal antibody or binding fragment of claim1, in which CDR-L1 has the substitutions Q4R+A11S.
 13. The monoclonalantibody or binding fragment of claim 1, in which CDR-L1 has thesubstitutions Q4G+A11G.
 14. The monoclonal antibody or binding fragmentof claim 1, in which CDR-L1 has the substitutions Q4H+A11S.
 15. Themonoclonal antibody or binding fragment of claim 1, in which CDR-L1 hasthe substitutions G5S+A11S.
 16. The monoclonal antibody or bindingfragment of claim 1, in which CDR-L2 has the substitution S3K.
 17. Themonoclonal antibody or binding fragment of claim 1, in which CDR-L2 hasthe substitution S3R.
 18. The monoclonal antibody or binding fragment ofclaim 1, in which CDR-L2 has the substitution T4H.
 19. The monoclonalantibody or binding fragment of claim 1, in which CDR-L2 has thesubstitution T4Q.
 20. The monoclonal antibody or binding fragment ofclaim 1, in which CDR-L2 has the substitution T4F.
 21. The monoclonalantibody or binding fragment of claim 1, in which CDR-L2 has thesubstitution T4W.
 22. The monoclonal antibody or binding fragment ofclaim 1, in which CDR-L2 has the substitution T4Y.
 23. The monoclonalantibody or binding fragment of claim 1, in which CDR-L2 has thesubstitution L5R.
 24. The monoclonal antibody or binding fragment ofclaim 1, in which CDR-L2 has the substitution L5K.
 25. The monoclonalantibody or binding fragment of claim 1, in which CDR-L2 has thesubstitution Q6R.
 26. The monoclonal antibody or binding fragment ofclaim 1, in which CDR-H1 has the substitution Y2H.
 27. The monoclonalantibody or binding fragment of claim 1, in which CDR-H1 has thesubstitution A3G.
 28. The monoclonal antibody or binding fragment ofclaim 1, in which CDR-H2 has the substitution T3N.
 29. The monoclonalantibody or binding fragment of claim 1, in which CDR-L2 furthercomprises the substitution S3N.
 30. The monoclonal antibody or bindingfragment of claim 1, in which CDR-L2 further comprises the substitutionT4V.
 31. The monoclonal antibody or binding fragment of claim 1, inwhich CDR-L2 further comprises the substitution Q6K.
 32. The monoclonalantibody or binding fragment of claim 1, in which CDR-H1 furthercomprises the substitution D1G.
 33. The monoclonal antibody or bindingfragment of claim 1 or claim 3, which has a K_(D) towards human TNF-αthat is 1.1-fold to 4-fold the K_(D) of an antibody having a V_(H)sequence of SEQ ID NO:2 and a V_(L) sequence of SEQ ID NO:4 as analyzedby BIAcore.
 34. The monoclonal antibody or binding fragment of claim 33,which has a K_(D) towards TNF-α that is 1.5-fold to 3-fold the K_(D) ofan antibody having a V_(H) sequence of SEQ ID NO:2 and a V_(L) sequenceof SEQ ID NO:4 as analyzed by BIAcore.
 35. An antibody-drug conjugatecomprising the monoclonal antibody or binding fragment of claim
 1. 36. Apharmaceutical composition comprising an anti-TNF-α antibody oranti-TNF-α binding fragment according to any one of claims 1 to 4 and apharmaceutically acceptable carrier.